Aprotinin analogs

ABSTRACT

The present invention relates to methods for producing aprotinin and analogs thereof in yeast, synthetic genes encoding such products, expression vectors and transformed yeast cells. The invention further relates to aprotinin analogs, particularly analogs with increased specific inhibitory activity and/or reduced nephrotoxicity compared to native aprotinin, as well as compositions comprising such analogs.

This application is a divisional application of co-pending application Ser. No. 08/084,718, filed Jun. 23, 1993, which is a continuation-in-part of application Ser. No. 08/024,925, filed Feb. 26, 1993, now abandoned, which is a continuation of application Ser. No. 07/466,408, filed Jun. 21, 1990, now abandoned. This application is also a continuation-in-part of application Ser. No. 07/598,737, filed Nov. 19, 1990, now U.S. Pat. No. 5,373,090, and a continuation-in-part of application Ser. No. 07/827,687, filed Jan. 29, 1992, now abandoned. This application also claims priority under 35 U.S.C. §120 to PCT application no. PCT/DK88/00138, filed Aug. 26, 1988, PCT application no. PCT/DK89/0096, filed Apr. 25, 1989, and PCT application on. PCT/DK91/0029, filed Oct. 1, 1991.

FIELD OF INVENTION

The present invention relates to methods for producing aprotinin and analogs thereof in yeast, synthetic genes encoding such products, expression vectors and transformed yeast cells. The invention further relates to aprotinin analogs, compositions comprising such analogs.

BACKGROUND OF THE INVENTION

Aprotinin (also known as bovine pancreatic trypsin inhibitor, BPTI) is a basic protein present in several bovine organs and tissues, such as the lymph nodes, pancreas, lungs, parotid gland, spleen and liver. It is a single-chain polypeptide of 58 amino acid residues with the following amino acid sequence

    __________________________________________________________________________     Arg                                                                               Pro                                                                               Asp                                                                               Phe                                                                               Cys                                                                               Leu                                                                               Glu                                                                               Pro                                                                               Pro                                                                               Tyr                                                                               Thr                                                                               Gly                                                                               Pro                                                                               Cys                                     Lys                                                                               Ala                                                                               Arg                                                                               Ile                                                                               Ile                                                                               Arg                                                                               Tyr                                                                               Phe                                                                               Tyr                                                                               Asn                                                                               Ala                                                                               Lys                                                                               Ala                                                                               Gly                                     Leu                                                                               Cys                                                                               Gln                                                                               Thr                                                                               Phe                                                                               Val                                                                               Tyr                                                                               Gly                                                                               Gly                                                                               Cys                                                                               Arg                                                                               Ala                                                                               Lys                                                                               Arg                                     Asn                                                                               Asn                                                                               Phe                                                                               Lys                                                                               Ser                                                                               Ala                                                                               Glu                                                                               Asp                                                                               Cys                                                                               Met                                                                               Arg                                                                               Thr                                                                               Cys                                                                               Gly                                     Gly                                                                               Ala                                                                               (SEQ ID NO: 1).                                                          __________________________________________________________________________

The amino acid chain is cross-linked by three disulphide bridges formed between Cys (5) and Cys (55), Cys (14) and Cys (38) and Cys (30) and Cys (51), respectively.

The isoelectric point of aprotinin is quite high (approximately 10.5). This is mainly caused by a relatively high content of the positively charged amino acids lysine and arginine. The three-dimensional structure of the aprotinin molecule is very compact which makes it highly stable against denaturation at high temperatures, or by acids, alkal is or organic solvents, or against proteolytic degradation (cf. B. Kassell, Meth. Enzym. 19, 1970, pp. 844-852).

Aprotinin is known to inhibit various serine proteases, including trypsin, chymotrypsin, plasmin and kallikrein, and is used therapeutically in the treatment of acute pancreatitis, various states of shock syndrome, hyperfibrinolytic hemorrhage and myocardial infarction (cf., for instance, J. E. Trapnell et al, Brit. J. Surg. 61, 1974, p. 177; J. McMichan et al., Circulatory shock 9, 1982, p. 107; L. M. Auer et al., Acta Neurochir. 49, 1979, p. 207; G. Sher, Am. J. Obstet. Gynecol. 129, 1977, p. 164; and B. Schneider, Artzneim.-Forsch. 26, 1976, p. 1606). Administration of aprotinin in high doses significantly reduces blood loss in connection with cardiac surgery, including cardiopulmonary bypass operations (cf., for instance, B. P. Bidstrup et al., J. Thorac. Cardiovasc. Surg. 97, 1989, pp. 364-372; W. van Oeveren et al., Ann Thorac. Surg. 44, 1987, pp. 640-645).

2.1. PRODUCTION OF APROTININ

Aprotinin, hereinafter referred to as "native aprotinin" can be extracted from various bovine organs or tissues, such as lung, pancreas and parotid glands. Extraction from animal tissues is a cumbersome process and requires large amounts of the bovine organ or tissue.

A gene for aprotinin has been fused to the coding sequence for E. coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter (Marks et al., 1986, J. Biol. Chem. 261: 7115-7118). Also, a synthetic gene encoding the protein sequence of Met-aprotinin has been cloned in an E. coli expression vector (yon Wilcken-Berman et al., 1986, EMBO J. 5: 3219-3225).

However, it has been found that a small protein such as aprotinin is likely to be degraded by the host proteases. Furthermore, it has been found to be difficult to establish correct disulphide bridges and correct folding in E. coli.

2.2. APROTININ ANALOGS

Certain aprotinin analogs are known, e.g. from U.S. Pat. No. 4,595,674 disclosing aprotinin analogs and derivatives wherein Lys(15) is replaced with Gly, Ala, Val, Leu, Ile, Met, Arg, L-α-butyric acid, L-norvaline, L-norleucine, dehydroalanine or L-homoserine. EP 238 993 discloses aprotinin analogs wherein Lys(15) is replaced with Arg, Val, Ile, Leu, Phe, Gly, Ser, Trp, Tyr or Ala, and wherein Met(52) is furthermore replaced with Glu, Val, Leu, Thr or Ser. EP 307 592 discloses aprotinin analogs wherein one or more of the amino acids in position 15, 16, 17, 18, 34, 39 and 52 are replaced with another amino acid residue. In position 17, the amino acid is preferably Leu, Arg, Ile or Val. Marks et al., 1987, Science 235: 1370-1373 describes mutants of aprotinin which are substituted by Ala or Thr in positions 14 and 38. It is reported that these mutants were expressed in E. coli and properly folded.

The known aprotinin analogs are claimed to have modified effects and efficacies towards different proteinages. For instance, aprotinin(15Val) has a relatively high selectivity for granulocyte elastase and an inhibitory effect on elastase and aprotinin (15Gly) has an outstanding antitrypsin activity and surprisingly inhibits kallikrein.

2.3. TOXICITY OF APROTININ

It has previously been described that after intravenous injection of native aprotinin in animals or human volunteers, the plasma level of the inhibitor decreases rather quickly owing to distribution in the extracellular fluid and subsequently accumulation in the kidneys (I. Trautschold et al., in K. Heinkel and H. Sch on (Eds.): Pathogenese, Diagnostik, Klinik und Therapie der Erkrankungen des Exokrinen Pankreas, Schattauer, Stuttgart, 1964, p. 289; E. Habermann et al., Med. Welt 24(29), 1973, pp. 1163-1167; H. Fritz et al., Hoppe-Seylers Z. Physiol. Chem. 350, 1969, pp. 1541-1550; and H. Kaller et al., Eur. J. Drug Metab. Pharmacokin. 2, 1978, pp. 79-85). Following glomerulus filtration, aprotinin is almost quantitatively bound to the brush border membrane of the proximal tubulus cells. Aprotinin is then reabsorbed into micropinocytic vesicles and phagosomes followed by a very slow degradation in phagolysosomes. This type of transport has been suggested to be representative for peptides in general (M. Just and E. Habermann, Navnyn-Scmiedebergs Arch. Pharmacol. 280, 1973, pp. 161-176; M. Just, Navnyn-Schmiedebergs Arch. Pharmacol. 287, 1975, pp. 85-95).

Macroscopic and histopathological examination following administration of aprotinin reveal changes in the renal tissues of rats, rabbits and dogs after repeated injections of relatively high doses of aprotinin (Bayer, Trasylol, Inhibitor of proteinase; E. Glaser et al. in "Verhandlungen der Deutschen Gesellschaft f ur Innere Medizin, 78. Kongress", Bergmann, M unchen, 1972, pp. 1612-1614). The nephrotoxicity (i.a. appearing in the form of lesions) observed for aprotinin might be ascribable to the accumulation of aprotinin in the proximal tubulus cells of the kidneys. This nephrotoxicity makes aprotinin less suitable for clinical purposes, in particular those requiring administration of large doses of the inhibitor (such as cardiopulmonary bypass operations).

OBJECTS OF THE INVENTION

It would be commercially advantageous to develop a method for producing high yields of properly folded aprotinin or aprotinin analogs in mature form.

Furthermore, it would be beneficial to obtain aprotinin analogs which have a more specific inhibitory effect towards certain serine proteases, such as elastase, kallikrein, t-PA, urokinase and coagulation factors, such as thrombin compared to native aprotinin.

It would also be commercially beneficial to produce aprotinin analogues with a reduced nephrotoxicity compared to native aprotinin.

SUMMARY OF THE INVENTION

The invention is directed to a method for producing high yields of aprotinin or analogs thereof. In a preferred embodiment, the aprotinin or aprotinin analog is produced in yeast by cultivation of a yeast strain containing a replicable expression vector containing a synthetic gene encoding aprotinin or analog thereof in a suitable nutrient medium followed by recovery of the aprotinin or analog thereof from the culture medium. The aprotinin produced by the present invention can be characterized by the following formula

    X-aprotinin(3-40)-Y.sub.n -Z.sub.m -aprotinin(43-58)

in which X means Arg-Pro, Pro or hydrogen, aprotinin(3-40) means the amino acid sequence from amino acid residue 3 to 40 in native aprotinin, Y may be Lys, or a non-basic amino acid residue, e.g., Ser, Thr or Ala, Z may be Arg, or a non basic amino acid residue, e.g. Ser, Thr or Ala, n and m are each 0 or 1, and aprotinin(43-58) means the amino acid sequence from amino acid residue 43 to 58 in native aprotinin.

The present invention is also directed to a DNA sequence comprising a synthetic gene encoding aprotinin or analog thereof.

The invention is also directed to a replicable expression vector comprising a DNA sequence or gene encoding aprotinin or analog thereof and a DNA sequence that allows for the expression of the aprotinin or analog thereof in yeast. The invention further provides a yeast strain transformed with such a vector.

The present invention is also related to novel aprotinin analogs. In one embodiment, the aprotinin analogs have a more specific inhibitory effect towards certain serine proteases such as elastase, kallikrein, t-PA, urokinase and coagulation factors such as thrombin than native protein.

In a specific embodiment, the aprotinin analog has the formula as set forth in the Sequence Listing as SEQ ID NO: 2. ##STR1## in which X₁ is Arg-Pro, Pro or hydrogen; X₂ and X₃ are independently any naturally occurring amino acid residue; X₄ and X₁₀ are both Cys; X₅ is Lys, Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Met, Trp, Tyr or Ala; X₆ is Ala or Gly; X₇ is Ala or Gly; X₈ is Ile, Leu, Met, Val or Phe; X₉ is any naturally occurring amino acid residue; X₁₁ is any naturally occurring amino acid residue; X₁₂ is Lys, Arg or Ser, provided that X₇ is Ala or Gly and each of X₂ -X₆ and X₈ -X₁₂ is different from the corresponding amino acid residue in native aprotinin.

In another specific embodiment, the aprotinin analog has the formula as set forth in the Sequence Listing as SEQ ID NO: 2. ##STR2## in which X₁ is Pro or hydrogen; X₂ and X₃ are independently any naturally occurring amino acid residue; X₄ and X₁₀ are both Cys; X₅ is Lys, Arg, Val, Thr, Ile, Leu, Phe, Gly, Ser, Met, Trp, Tyr or Ala; X₆ is Ala or Gly; X₇ is any naturally occuring amino acid residue; X₈ is Ile, Leu, Met, Val or Phe; X₉ is any naturally occurring amino acid residue; X₁₁ is any naturally occurring amino acid residue; X₁₂ is Lys, Arg or Ser, provided that X₁ is Pro or hydrogen and at least one of the amino acid residues X₂ to X₉ is different from the corresponding amino acid residue in native aprotinin.

In another embodiment, the present invention further relates to an aprotinin analog with reduced nephrotoxicity compared to native aprotinin. In a preferred embodiment, such an analog has a reduced net positive charge and reduced stability compared to native aprotinin. To provide a reduced positive net charge, at least one positively charged amino acid residue outside the protease-binding site is removed or replaced with a neutral or negatively charged amino acid residue, and/or at least one negatively charged amino acid residue is inserted or added, and/or wherein at least one neutral amino acid residue is replaced with a negatively charged amino acid residue. To provide reduced stability, one or more amino acid residues are deleted, added or replaced with one or more other amino acid residues. In a specific embodiment, the aprotinin analog has the following formula

    X'-aprotinin(3-40)-Y'.sub.n -Z'.sub.m -aprotinin(43-58)

in which X' means Pro or hydrogen, aprotinin(3-40) means the amino acid sequence from amino acid residue 3 to 40 in native aprotinin, Y' is Lys or a non-basic amino acid residue, Z' may be Arg or a non-basic amino acid residue with the proviso that at least Y' or Z' is a non-basic amino acid residue, n and m are each 0 or 1, and aprotinin(43-58) means the amino acid sequence from amino acid residue 43 to 58 in native aprotinin.

In the present context, the term "reduced positive net charge" is understood to include analogues with a lower positive net charge than that of native aprotinin (which has a positive net charge of +6) as well as with no net charge or a negative net charge. It should be noted that the net charge of aprotinin may vary according to pH, and that the terms "positive net charge", "negative net charge", "positively charged" or "negatively charged" are used about the charge of the molecule at a neutral pH.

The term "protease-binding site" is intended to indicate the amino acid residues which are important for protease inhibition, i.e. the amino acid residues which are in intimate contact with the protease by binding to amino acid residues at or close to the active site of the enzyme. These are currently understood to include (and are, in the present context defined as) the amino acid residues in position 12-18 and 34-39 (cf. H. Fritz and G. Wunderer, Artzneim.-Forsch. 33(1), 1983, p. 484). It is preferred to remove, insert or replace amino acid residues outside the protease-binding site only in order to avoid substantially changing the protease inhibition profile of the analogue of the invention compared to that of native aprotinin.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be further illustrated by reference to the accompanying drawings in which:

FIG. 1 shows a synthetic gene encoding aprotinin(3-58).

FIG. 2 illustrates the construction of plasmids pKFN374 and pKFN375.

FIG. 3 illustrates the construction of plasmid pMT 636.

FIG. 4 illustrates the construction of plasmids pKFN414 and pKFN416.

FIG. 5 shows a synthetic gene encoding aprotinin (3-58; 42 Ser).

FIG. 6 illustrates the construction of the plasmid pKFN 306.

FIG. 7 illustrates the construction of the plasmid pKFN 504.

FIG. 8 illustrates the construction of the plasmid pMT 636.

FIG. 9A illustrates the inhibition of plasma kallikrein by native aprotinin and by the aprotinin analogs KFN 396 and KFN 399.

FIG. 9B illustrates the inhibition of plasma kallikrein by native aprotinin and by the aprotinin analogs KFN 396, KFN 772 and KFN 773.

FIG. 10 shows the construction of a synthetic aprotinin gene from oligonucleotide sequences.

FIG. 11 shows the construction of plasmid pKFN-1503.

FIG. 12 is a graph showing the inhibitory activity in urine and kidneys after administration of aprotinin analogues with different net charges and thermal stability.

FIG. 13 shows the inhibitory activity in urine 3 hours after administration of aprotinin analogues with different net charges.

FIG. 14 shows the inhibitory activity in kidneys 3 hours after administration of aprotinin analogues with a different thermal stability.

FIG. 15 shows the accumulation of inhibitory activity in kidneys after administration of aprotinin analogues with different thermal stability. The accumulation index is calculated as the inhibitory activity after 3 hours divided by the inhibitory activity after 1 hour.

FIG. 16 shows a synthetic gene encoding aprotinin(1-58).

FIG. 17 shows a synthetic gene encoding aprotinin(1-58,42 Ser).

DETAILED DESCRIPTION OF THE INVENTION

The invention is related to a method for producing high yields of aprotinin or analogs thereof, as well as genes encoding aprotinin and analogs thereof, vectors comprising such genes, and host cells capable of expressing the genes. The invention is directed to novel aprotinin analogs, specifically analogs having a more specific inhibitory effect towards certain serine proteases and analogs having a reduced nephrotoxicity compared to native aprotinin, as well as pharmaceutical compositions comprising such novel analogs.

6.1. PRODUCTION OF APROTININ AND APROTININ ANALOGS

Aprotinin and the aprotinin analogs of the present invention may be obtained by recombinant DNA methods known in the art exemplified below and described in the examples herein. A DNA construct is prepared comprising a gene encoding aprotinin or an analog thereof.

The DNA construct may be prepared synthetically by established standard methods, e.g. the phosphoramidite method described by S. L. Beaucage and M. H. Caruthers, Tetrahedron Letters 22, 1981, pp. 1859-1869, or the method described by Matthes et al., EMBO Journal 3, 1984, pp. 801-805. According to the phosphoramidite method, oligonucleotides are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in suitable vectors.

Alternatively, it is possible to use genomic or cDNA coding for native aprotinin (e.g. obtained by screening a genomic or cDNA library using synthetic oligonucleotide probes) and modified at one or more sites corresponding to the site(s) at which it is desired to introduce amino acid substitutions, e.g. by site-directed mutagenesis using synthetic oligonucleotides encoding the desired amino acid sequence for homologous recombination in accordance with well-known procedures.

A vector which comprises the DNA construct and is capable of expressing the gene encoding the aprotinin or analog thereof in a host cell is any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

In a preferred embodiment, the vector is capable of replicating in yeast cells. As will be detailed in the Examples herein, aprotinin and aprotinin analogs can surprisingly be produced in high yields with correctly positioned disulphide bridges by cultivation of a yeast cell transformed with a gene encoding such products.

The gene encoding aprotinin or an aprotinin analog in the vector should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the aprotinin or aprotinin analog are the SV 40 promoter (Subramani et al., Mol. Cell Biol. 1, 1981, pp. 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222, 1983, pp. 809-814) or the adenovirus 2 major late promoter. Suitable promoters for use in yeast host cells include promoters from yeast glycolytic genes (Hitzeman et al., J. Biol. Chem. 255, 1980, pp. 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or alcohol dehydrogenase genes (Young et al., in Genetic Engineering of Microorganisms for Chemicals (Hollaender et al, eds.), Plenum Press, New York, 1982), or the TPI1 (U.S. Pat. No. 4,599,311) or ADH2-4c (Russell et al., Nature 304, 1983, pp. 652-654 ) promoters. Suitable promoters for use in filamentous fungus host cells are, for instance, the ADH3 promoter (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099) or the tpiA promoter.

For secretion purposes, the DNA sequence encoding the desired aprotinin or aprotinin analog may be fused to a DNA sequence encoding a signal and leader peptide sequence. The signal and leader peptides are cleaved off by the transformed microorganism during the secretion of the expressed protein product from the cells ensuring a more simple isolation procedure of the desired product. A well suited leader peptide system for yeast is the yeast MFal leader sequence or a part thereof (Kurjan, J. and Herskowitz, I., Cell 30 (1982) 933-943) or a leader described in Danish patent application No. 4638/87. However, any signal- or leader-sequence which provides for secretion in yeast may be employed and the present invention is not contemplated to be restricted to a specific secretion system.

The DNA sequence encoding the aprotinin analogue of the invention may also be operably connected to a suitable terminator, such as the human growth hormone terminator (Palmiter et al., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op. cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector may further comprise elements such as polyadenylation signals (e.g. from SV 40 or the adenovirus 5 Elb region), transcriptional enhancer sequences (e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. the ones encoding adenovirus VA RNAs).

The vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such a sequence (when the host cell is a mammalian cell) is the SV 40 origin of replication, or (when the host cell is a yeast cell) the yeast plasmid 2μ replication genes REP 1-3 and origin of replication. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or one which confers resistance to a drug, e.g. neomycin, hygromycin or methotrexate, or the Schizosaccharomyces pombe TPI gene (described by P. R. Russell, Gene 40, 1985, pp. 125-130.

The procedures used ligate the DNA sequences coding for the aprotinin or aprotinin analog, the promoter, and the terminator, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989).

The host cell into which the vector of the invention is introduced may be any cell which is capable of producing aprotinin or an aprotinin analog and is preferably a eukaryotic cell, such as a mammalian, yeast or fungal cell.

Examples of suitable mammalian cell lines are the COS (ATCC CRL 1650), BHK (ATCC CRL 1632, ATCC CCL 10) or CHO (ATCC CCL 61) cell lines. Methods of transfecting mammalian cells and expressing DNA sequences introduced in the cells are described in e.g. Kaufman and Sharp, J. Mol. Biol, 159, 1982, pp. 601-621; Southern and Berg, J. Mol. Appl. Genet, 1, 1982, pp. 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA 79, 1982, pp. 422-426; Wigler et al., Cell 14, 1978, p, 725; Corsaro and Pearson, Somatic Cell Genetics 7, 1981, p. 603, Graham and van der Eb, Virology 52, 1973, p. 456; and Neumann et al., EMBO J. 1, 1982, pp. 841-845.

In a preferred embodiment, the yeast organism used as the host cell according to the invention may be any yeast organism which, on cultivation, produces large quantities of the aprotinin analog of the invention. Examples of suitable yeast organisms are strains of the yeast species Saccharomyces cerevisiae, Saccharomyces kluyveri, Schizosaccharomyces pombe or Saccharomyces uvarum. The transformnation of yeast cells may for instance be effected by protoplast formation followed by transformation in a manner known per se. The methods for transformation of yeast and cultivation of transformed yeast strains that can be used in the practice of the invention are those known in the art such as those described in the above mentioned EP patent application NOS. 0163529A and 0189998A.

Alternatively, fungal cells may be used as host cells of the invention. Examples of suitable fungal cells are cells of filamentous fungi, e.g. Aspergillus spp. or Neurospora spp., in particular strains of Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp. for the expression of proteins is described in, e.g., EP 272 277.

The medium used to cultivate the cells may be any conventional medium suitable for growing mammalian cells or yeast organisms, depending on the choice of host cell. The aprotinin or aprotinin analog will be secreted by the host cells to the growth medium and may be recovered therefrom by conventional procedures including separating the cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulfate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography or affinity chromatography, or the like.

6.2. ANALOGS HAVING A MORE SPECIFIC INHIBITORY EFFECT TOWARDS CERTAIN SERINE PROTEASES

The present aprotinin analogs may be represented by the formula as set forth in the Sequence Listing as SEQ ID NO: 2 and in Formulae I and II (see Section 4, supra).

According to a more narrow aspect, the present aprotinin analogs may be represented by the following formula set forth in the Sequence Listing as SEQ ID NO: 3: ##STR3## in which X₁, X₅, X₆, X₇, X₈, X₉, X₁₁ and X₁₂ are as defined above for SEQ ID NO: 2, at least one of the amino acid residues X₅ to X₉, preferably X₆ to X₉ being different from the corresponding amino acid residue in native aprotinin.

According to an even narrower aspect the aprotinin analogues may be represented by the following formula set forth in the Sequence Listing as SEQ ID NO: 4: ##STR4## in which X₁, X₆, X₇, X₈, X₉, X₁₁ and X₁₂ are as defined above for SEQ ID NO: 2, at least one of the amino acid residues X₆ to X₉ being different from the corresponding amino acid residue in native aprotinin.

In the sequence represend by formula I or II above, X₁ is preferably hydrogen; X₂ is preferably Gly; X₃ is preferably Pro; X₅ is preferably Lys or Arg; X₆ is preferably Ala; X₇ is preferably Ala; X₉ is preferably Ile; X₉ is preferably Ile; X₁₁ is preferably Lys; and/or X₁₂ is preferably Arg or Ser.

Examples of preferred aprotinin analogs according to the present invention are aprotinin (3-58; 17 Ala+42 Ser; SEQ ID NO: 5) which lacks the first two amino acid residues of native aprotinin and has Ala substituted for Arg in position 17 and Ser substituted for Arg in position 42; aprotinin (3-58; 17 Ala+19 Glu+42 Ser; SEQ ID NO: 6) which lacks the first two amino acid residues of native aprotinin and has Ala substituted for Arg in position 17, Glu substituted for Ile in position 19 and Ser substituted for Arg in position 42; and aprotinin (3-58; 15 Arg+17 Ala+42 Ser; SEQ ID NO: 7) which lacks the first two amino acid residues of native aprotinin and has Arg substituted for Lys in position 15, Ala substituted for Arg in position 17 and Ser substituted for Arg in position 42, respectively.

Further examples of aprotinin analogs to the present invention are:

Aprotinin (3-58; 17 Ala) (SEQ ID NO: 8)

Aprotinin (3-58; 17 Ala+19 Glu) (SEQ ID NO: 9)

Aprotinin (3-58; 15 Arg+17 Ala) (SEQ ID NO: 10)

Aprotinin (17 Ala+42 Ser) (SEQ ID NO: 11)

Aprotinin (15 Arg+17 Ala+42 Ser) (SEQ ID NO: 12)

Aprotinin (17 Ala) (SEQ ID NO: 13)

Aprotinin (17 Ala+19 Glu) (SEQ ID NO: 14)

Aprotinin (15 Arg+17 Ala) (SEQ ID NO: 15)

6.3. APROTININ ANALOGS WITH REDUCED NEPHROTOXICITY

The invention is also directed to aprotinin analogs having reduced nephrotoxicity compared to native aprotinin. In a specific embodiment, such analogs have a reduced positive charge and reduced stability.

According to the invention, any of the positively charged amino acid residues outside the protease-binding site might be replaced with either the negatively charged amino acid residues Glu or Asp or with any one of the neutral amino acid residues Ala, Cys, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Glu, Ser, Thr, Val, Trp or Tyr. However, in order to avoid inactive analogs or analogs with an inappropriate three-dimensional structure arising from an undesired folding of the molecule, it is preferred to select substitutions which are identical to amino acid residues in corresponding positions of other protease inhibitors or in domains of larger structures exhibiting a high degree of homology to native aprotinin. In other words, the selection of substituent amino acid residues is preferably based on an analysis of molecules which are homologous to aprotinin. It should be noted that, concomitantly with the amino acid substitution(s) directly contributing to a reduction in the positive net charge, one or more other amino acid substitutions may be carried out which do not in themselves result in a reduced positive net charge, but which may be required in order to produce an active analogue with an appropriate three-dimensional structure.

Accordingly, in a more specific aspect, the present invention relates to an aprotinin analog having the following formula set forth in the Sequence Listing as SEQ ID NO: 16:

    __________________________________________________________________________     X.sub.1                                                                           Asp                                                                               Phe                                                                               Cys                                                                               Leu                                                                               Glu                                                                               Pro                                                                               Pro                                                                               X.sub.2                                                                           Thr                                                                               Gly                                                                               Pro                                                                               Cys                                                                               Lys                                                                               Ala                                  Arg                                                                               Ile                                                                               Ile                                                                               X.sub.3                                                                           Tyr                                                                               Phe                                                                               Tyr                                                                               X.sub.4                                                                           Ala                                                                               X.sub.5                                                                           Ala                                                                               Gly                                                                               Leu                                                                               Cys                                                                               X.sub.6                                                                           Thr                               Phe                                                                               Val                                                                               Tyr                                                                               Gly                                                                               Gly                                                                               Cys                                                                               Arg                                                                               X.sub.7                                                                           X.sub.8                                                                           X.sub.9                                                                           Asn                                                                               X.sub.10                                                                          Phe                                                                               X.sub.11                                                                          Ser                                                                               Ala                               Glu                                                                               Asp                                                                               Cys                                                                               Met                                                                               X.sub.12                                                                          Thr                                                                               Cys                                                                               Gly                                                                               Gly                                                                               Ala                                                 __________________________________________________________________________

wherein

X₁ is an amino acid residue selected from the group consisting of Tyr, Glu, Asp, Ser, Thr, Ala and Val,

X₂ is an amino acid residue selected from the group consisting of Tyr, Glu, Asp, Ser, Thr, Ala and Val,

X₃ is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Leu, Ser, Ala, Gln and Thr,

X₄ is an amino acid residue selected from the group consisting of Asn, Glu and Asp,

X₅ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Thr, Val, Ala, Ser, Phe, Gln and Gly,

X₆ is an amino acid residue selected from the group consisting of Gln, Glu, Asp, Val and Ala,

X₇ is an amino acid residue selected from the group consisting of Ala, Asp, Glu and Gly,

X₈ is an amino acid residue selected from the group consisting of Lys, Glu, AsP, Asn, Ser, Thr and Ala,

X₉ is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Ser, Asn, Leu, Gly, Gln, Met and Thr,

X₁₀ is an amino acid residue selected from the group consisting of Asn, Glu and Asp,

X₁₁ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Leu, Tyr, Ala, Val, Thr, Ser, Pro, His and Ile, and

X₁₂ is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Gln, Ala, Asn, His, Gly, Ser and Thr,

with the proviso that at least one of the amino acid residues X₁ -X₁₂ is different from the corresponding amino acid residue of native aprotinin.

Apart from these internal substitutions in the aprotinin molecule, it may be possible to add a peptide containing one or more negatively charged amino acid residues (i.e. Glu or Asp) at the N- or C-terminal end of the aprotinin molecule in order to provide the requisite reduction in the positive net charge. It may further be possible, in order to provide a reduction in the stability of the molecule, to add one or more neutral amino acid residues to the N- or C-terminal end of the molecule. Such additions may be done either to the native aprotinin molecule or in addition to other modifications as indicated above.

If it is desired to change the protease-inhibition properties of the aprotinin analogue apart from reducing its nephrotoxicity, it is possible additionally to modify the analog in the protease-binding site. For instance, it has previously been demonstrated (cf. H. R. Wenzel and H. Tschesche, Angew. Chem. Internat. Ed. 20, 1981, p. 295) that aprotinin(1-58, Val15) exhibits a relatively high selectivity for granulocyte elastase and an inhibitory effect on collagenase, aprotinin (1-58, Ala15) has a weak effect on elastase, and aprotinin (1-58, Gly15) exhibits an outstanding antitrypsin activity and surprisingly also inhibits kallikrein. Furthermore, it may be possible to modify the inhibitory effect of aprotinin concomitantly with reducing the positive net charge by replacing one or more positively charged amino acids in the protease-binding site by neutral or negatively charged amino acid(s).

Thus, the present invention further relates to an aprotinin analog having the following formula set forth in the Sequence Listing as SEQ ID NO: 17:

    __________________________________________________________________________     X.sub.1                                                                           Asp                                                                               Phe                                                                               Cys                                                                               Leu                                                                               Glu                                                                               Pro                                                                               Pro                                                                               X.sub.2                                                                           Thr                                                                               Gly                                                                               Pro                                                                               Cys                                                                               X.sub.13                                                                          X.sub.14                                                                          X.sub.15                          X.sub.16                                                                          X.sub.17                                                                          X.sub.3                                                                           Tyr                                                                               Phe                                                                               Tyr                                                                               X.sub.4                                                                           Ala                                                                               X.sub.5                                                                           Ala                                                                               Gly                                                                               Leu                                                                               Cys                                                                               X.sub.6                                                                           Thr                                                                               Phe                               X.sub.18                                                                          Tyr                                                                               X.sub.19                                                                          Gly                                                                               Cys                                                                               X.sub.20                                                                          X.sub.7                                                                           X.sub.8                                                                           X.sub.9                                                                           Asn                                                                               X.sub.10                                                                          Phe                                                                               X.sub.11                                                                          Ser                                                                               Ala                                                                               Glu                               Asp                                                                               Cys                                                                               Met                                                                               X.sub.12                                                                          Thr                                                                               Cys                                                                               Gly                                                                               Gly                                                                               Ala                                                    __________________________________________________________________________

wherein X₁ -X₁₂ are as defined above,

X₁₃ is an amino acid residue selected from the group consisting of Lys, Arg, Glu, Leu, Met, Tyr and Phe,

X₁₄ is an amino acid residue selected from the group consisting of Ala and Gly,

X₁₅ is an amino acid residue selected from the group consisting of Arg, Ala, Gly, Lys, Leu, Met, Phe, Tyr, Ile and Asn,

X₁₆ is an amino acid residue selected from the group consisting of Ile, Met, Leu, Phe, Thr and Glu,

X₁₇ is an amino acid residue selected from the group consisting of Ile, Leu, Lys, Gln, Glu, Ser, Arg, Thr and Asn,

X₁₈ is an amino acid residue selected from the group consisting of Val, Thr, Leu, Ser, Tyr, Gln, His, Pro, Phe, Asn, Ile and Lys,

X₁₉ is an amino acid residue selected from the group consisting of Gly, Thr and Ser, and

X₂₀ is an amino acid residue selected from the group consisting of Gln, Lys, Met, Asn, Leu, Gly and Glu,

with the proviso that at least one of the amino acid residues X₁ -X₁₂ and at least one of the amino acid residues X₁₃ -X₁₀ are different from the corresponding amino acid residue of native aprotinin.

Examples of currently favored aprotinin analogs of the general formula (SEQ ID NO: 16) include an analog wherein X₁ is Glu-Pro, X₅ is Glu, X₈ is Glu, X¹¹ is Glu, and X₂, X₃, X₄, X₆, X₇, X₉, X₁₀ and X₁₂ are as in the native aprotinin sequence; or wherein X₁ is Glu-Pro, X₉ is Glu, X₁₁ is Glu, and X₂, X₃, X₄, X₆, X₇, X₈, X₁₀ and X₁₂ are as in the native aprotinin sequence; or wherein X₉ is Glu, X₁₁ is Glu, and X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₂ are as in the native aprotinin sequence; or wherein X₂ is Ser, X₄ is Asp, X₅ is Thr, X₆ is Glu, X₈ is Asn, X₁₂ is Glu, and X₁, X₃, X₇, X₉, X₁₀, and X₁₁ are as in the native aprotinin sequence; or wherein X₂ is Ser, X₃ is Leu, X₇ is Gly, X₈ is Asn, X₉ is Gly, X₁₀ is Gln, X₁₁ is Tyr, and X₁, X₄, X₅, X₆, and X₁₂ are as in the native aprotinin sequence; or wherein X₁ is a peptide bond, X₉ is Ser, X₁₁ is Glu, and X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₂ are as in the native aprotinin sequence or wherein X₁ is a peptide bond, X₉ is Ser, X₁₁ is Ala, and X₂, X₃, X₄, X₅, X₆, X₇, X₈ and X₁₀ and X₁₂ are as in the native aprotinin sequence; or wherein X₁ is a peptide bond, X₂ is Ser, X₄ is Asp, X₅ is Thr, X₆ is Glu, X₈ is Asn, X₁₂ is Glu, and X₃, X₇, X₉, X₁₀ and X₁₁ are as in the native aprotinin sequence; or wherein X₁ is a peptide bond, X₄ is Asp, X₅ is Thr, X₆ is Glu, X₁₂ is Glu, and X₂, X₃, X₇, X₈, X₉, X₁₀ and X₁₁ are as in the native aprotinin sequence; or wherein X₁ is a peptide bond, X₂ is Ser, X₇ is Gly, X₈ is Asn, X₉ is Gly, X₁₂ is Glu, and X₃, X₄, X₅, X₆, X₁₀ and X₁₁ are as in the native aprotinin sequence; or wherein X₁ is a peptide bond, X₉ is Ser, X₁₂ is Glu, and X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₁ are as in the native aprotinin sequence; or wherein X₁ is a peptide bond, X₉ is Glu, X₁₂ is Glu, and X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₁ are as in the native aprotinin sequence; or wherein X₁ is a peptide bond, X₅ is Glu, X₉ is Ser, X₁₂ is Glu, and X₂, X₃, X₄, X₆, X₇, X₈, X₁₀ and X₁₁ are as in the native aprotinin sequence; or wherein X₁ is a peptide bond, X₅ is Glu, X₉ is Glu, X₁₂ is Glu, and X₂, X₃, X₄, X₆, X₇, X₈, X₁₀ and X₁₁ are as in the native aprotinin sequence.

In another embodiment, the aprotinin analogs of the present invention have the formula

    X'-aprotinin(3-40)-Y'.sub.n -Z'.sub.m -aprotinin(43-58)

in which X' means Pro or hydrogen, aprotinin(3-40) means the amino acid sequence from amino acid residue 3 to 40 in native aprotinin, Y' is Lys or a non-basic amino acid residue, Z' may be Arg or a non-basic amino acid residue with the proviso that at least Y' or Z' is a non-basic amino acid residue, n and m are each 0 or 1, and aprotinin(43-58) means the amino acid sequence from amino acid residue 43 to 58 in native aprotinin.

6.4. USES FOR APROTININ ANALOGS

The analogs of the present invention as a result of their inhibition of human serine proteases may be used to treat acute pancreatitis, inflammation, thrombocytopenia, preservation of platelet function, organ preservation, wound healing, shock (including shock lung) and conditions involving hyperfibrinolytic hemorrhage. A high dose of aprotinin is indicated during and after cardiopulmonary bypass operations. The aprotinin analogs of the present invention having a lower nephrotoxicity is of particular interest for this application and possibly in other surgery involving a major loss of blood, as is the possibly reduced risk of causing anaphylactoid response due to the lower positive net charge of the analog.

The analogs of the present invention may be formulated in a pharmaceutical composition with an acceptable carrier. The pharmaceutical carriers may be such physiologically compatible buffers as Hank's or Ringer's solution, physiological saline, a mixture consisting of saline and glucose, and heparinized sodium-citrate-citric acid-dextrose solution. The aprotinin analogs of the present invention produced by the methods of the present invention can be mixed with colloidal-like plasma substitutes and plasma expanders such as linear polysaccharides (e.g. dextran), hydroxyethyl starch, balanced fluid gelatin, and other plasma proteins. Additionally, the aprotinin analogs may be mixed with water soluble, physiologically acceptable, polymeric plasma substitutes, examples of which include polyvinyl alcohol, poly(ethylene oxide), polrvinylpyrrolidone, and ethylene oxide-polypropylene glycol condensates. Techniques and formulations for administering the compositions comprising the aprotinin analogs generally may be found in Remington's Pharmaceutical Sciences, Meade Publishing Col., Easton, Pa., latest edition.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES 7.1. EXAMPLE 1: PRODUCTION OF APROTININ(3-58)

A sequence encoding aprotinin (3-58) was constructed from a number of oligonucleotides by ligation.

The oligonucleotides were synthesized on an automatic DNA synthesizer using phosphoramidite chemistry on a controlled pore glass support (S. L. Beaucage and M. H. Caruthers (1981) Tetrahedron Letters 22, 1859-1869).

The following 10 oligonucleotides were synthesized: ##STR5##

5 duplexes A-E formed from the above 10 oligonucleotides as shown in FIG. 1.

20 pmole of each of the duplexes A-E was formed from the corresponding pairs of the above oligonucleotides by heating for 5 min. at 90° C. followed by cooling to room temperature over a period of 75 minutes. The five duplexes were mixed and treated with T4 ligase. The synthetic gene was isolated as a 176 bp band after electrophoresis of the ligation mixture on a 2% agarose gel. The obtained synthetic gene is shown in FIG. 1 and set forth in the Sequence Listing as SEQ ID NOS: 28 AND 29. The synthetic gene was ligated to a 330 bp EcoRI-HgaI fragment from plasmid pKFN9 coding for MFαl signal and leader sequence(1-85) and to the large EcoRI-XbaI fragment from pUC19. The construction of pKFN9 containing a HgaI site immediately after the MFαl leader sequence is described in EP application No. 0214826.

The ligation mixture was used to transform a competent E. coli strain (r⁻, m⁺) selecting for ampicillin resistance. Sequencing of a ³² P-XbaI-EcoRI fragment (Maxam, A. and Gilbert, W., Methods Enzymol. 65 (1980) 499-560) showed that plasmids from the resulting colonies contained the correct DNA-sequence for aprotinin(3-58).

One plasmid pKNF305 was selected for further use. The construction of plasmid pKFN305 is illustrated in FIG. 2. DKFN305 was cut with EcoRI and XbaI and the 0.5 kb fragment was ligated to the 9.5 kb NcoI-XbaI fragment from pMT636 and the 1.4 kb NcoI-EcoRI fragment from pMT636, resulting in plasmid pKFN374 (see FIG. 2). Plasmid pMT636 was constructed from pMT608 after deletion of the LEU-2 gene and from pMT479 (see FIG. 3). pMT608 is described in EP application No. 1956911. pMT479 is described in EP application No. 163529. pMT479 contains the Schizo. pombe TPI gene (POT), the S. cerevisiae triosephosphate isomerase promoter and terminator, TPI_(P) and TPI_(T) (Alber, T. and Kawasaki, G. J. Mol. Appl. Gen. 1 (1982) 419-434). Plasmid pKFN374 contains the following sequence

    TPI.sub.P -MFαl-signal-leader(1-85)-aprotinin(3-58)-TPI.sub.T

where MFαl is the S. cerevisiae mating factor alpha 1 coding sequence (Kurjan, J. and Herskowitz, I., Cell 30, (1982) 933-943), signal-leader(1-85) means that the sequence contains the first 85 amino acid residues of the MFαl signal-leader sequence and aprotinin(3-58) is the synthetic sequence encoding an aprotinin derivative lacking the first two amino acid residues.

S. cerevisiae strain MT663 (E2-7B XE11-36 a/α, Δ tpi Δ tpi, pep 4-3/pep 4-3) was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an O.D. at 600 nm of 0.6.

100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of a solution containing 1.2M sorbitol, 25 mM Na₂ EDTA pH=8.0, and 6.7 mg/ml dithiothreitol. The suspension was incubated at 30° C. for 15 minutes, centrifuged and the cells resuspended in 10 ml of a solution containing 1.2M sorbitol, 10 mM Na₂ EDTA, 0.1M sodium citrate, pH=5.8, and 2 mg Novozym® 234. The suspension was incubated at 30° C. for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2M sorbitol and 10 ml of CAS (1.2M sorbitol, 10 mM CaCl₂, 10 mM Tris HCl (Tris=Tris (hydroxymethyl) aminomethane, pH=7.5) and resuspended in 2 ml of CAS. For transformation 0.1 ml of CAS-resuspended cells were mixed with approximately 1 μg of plasmid pKFN374 and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4000, 10 mM CaCl₂, 10 mM Tris HCl, pH=7.5) was added and the mixture left for further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2M sorbitol, 33% v/v YPD, 6.7 mM CaCl₂, 14 Ng/ml leucine) and incubated at 30° C. for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2M sorbitol. Then, 6 ml of top agar (the SC medium of Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) containing 1.2M sorbitol plus 2.5% agar) at 52° C. was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium. Transformant colonies were picked after 3 days at 30° C., reisolated and used to start liquid cultures. One such transformant KFN322 was chosen for further characterization.

Yeast strain KFN322 was grown on YUPD medium (1% yeast extract, 2% peptone (from Difco Laboratories), and 2% glucose). A 10 ml culture of the strain was shaken at 30° C. to an O.D. at 600 nm of 32. After centrifugation, the supernatant was analyzed by FPLC ion exchange chromatography. The yeast supernatant was filtered through a 0.22 μm Millex® GV filter unit and 1 ml was applied on a MonoS cation exchange column (0.5×5 cm) equilibrated with 20 mM Bicine, pH 8.7. After wash with equilibration buffer the column was eluted with a linear NaCl gradient (0-1M) in equilibration buffer. Trypsin inhibitor activity was quantified in the eluted fractions by spectrophotometric assay and furthermore by integration of absorption at 280 nm from

    E.sup.1%.sub.280 (aprotinin)=8.3

The yield was about 3 mg/liter of aprotinin(3-58).

For amino acid analysis and N-terminal sequencing, the yeast supernatant (7 ml) was adjusted to pH 8.7 with 0.1M NaOH and filtered (0.22 μm). The effluent from a Q-Sepharose anion exchange column (1×4 cm) equilibrated with 20 mM Bicine, pH 8.7 was applied to a MonoS cation exchange column (0.5×5 cm). The cation exchange chromatography was carried out as described above. Concentration of the gradient eluted aprotinin (3-58) was accomplished by rechromatography on MonoS and elution with a steep NaCl-gradient. The collected fractions were further concentrated by vacuum centrifugation to about 100 μl and applied to a RP-HPLC column (Vydac C4, 4.6×250 mm). Elution was carried out with CH₃ CN gradient in 0.1% TFA. The collected fractions were concentrated to about 100 μl by vacuum centrifugation and samples were taken for N-terminal sequencing and amino acid analysis.

By N-terminal sequencing the following sequence was found ##STR6## confirming that the N-terminal end is correct.

The amino acid analysis is shown in following Table 1. From this table it appears that the product has the expected amino acid composition, i.e., less Arg and Pro. The slightly lowered content of Ile can most probably be ascribed incomplete hydrolysis of Ile(18)-Ile(19) (this is well known in the art). Also, Pro and Arg is slightly higher than expected. This is, however, also seen with aprotinin itself (Table 1, second column).

When compared by the above mentioned method of Erlanger et al. the specific activity of aprotinin(3-58) was found to be identical within the experimental error with the specific activity of native aprotinin.

                  TABLE 1                                                          ______________________________________                                                                     Aprotinin                                                                             Aprotinin                                   Amino  Aprotinin  Aprotinin (3-58) (3-58,42 Ser)                               Acid   (Theoretical)                                                                             (Found)   (Found)                                                                               (Found)                                     ______________________________________                                         Asx    5          5.00      4.96   5.02                                        Thr    3          2.86      2.83   2.85                                        Ser    1          0.94      0.97   1.78                                        Glx    3          3.04      3.01   3.02                                        Pro    4          4.18      3.15   3.19                                        Gly    6          5.95      6.00   5.99                                        Ala    6          5.85      5.93   6.01                                        Cys    6          5.20      5.03   5.41                                        Val    1          0.99      0.98   0.98                                        Met    1          0.83      0.85   0.96                                        Ile    2          1.39      1.41   1.50                                        Leu    2          1.97      1.98   2.03                                        Tyr    4          3.84      3.80   3.82                                        Phe    4          3.98      3.92   3.96                                        Lys    4          3.92      4.02   3.93                                        Arg    6          6.39      5.20   4.28                                        Total  58         56.33     54.04  54.73                                       ______________________________________                                    

7.2. EXAMPLE 2: PRODUCTION OF APROTININ (3-58, 42 Ser)

A synthetic gene for aprotinin(3-58, 42 Ser; SEQ ID NO: 29) was constructed as described in Example 1 (Section 7.1., supra). With the purpose of substituting Arg(42) with Ser the following oligonucleotides VIIa and VIIIa were used instead of VII and VIII: ##STR7##

The obtained synthetic gene is shown in FIG. 5 (SEQ ID NOS: 33 and 34). This gene fused to the MFαl signal-leader(1-85) sequence was cloned in a pUC19 derived plasmid pKFN306 (see FIG. 6).

By following the procedure of Example 1a plasmid pKFN375 was obtained containing the following construction

    TPI.sub.P -MFαl-signal-leader(1-85)-aprotinin(3-58, 42 Ser)-TPI.sub.T

where aprotinin(3-58, 42 Ser) is the synthetic gene encoding an aprotinin derivative lacking the first two amino acid residues and containing a Ser instead of Arg in position 42.

Yeast strain MT663 was transformed with plasmid pKFN375 as described above, and culturing of the transformed strain KFN324 gave about 12 mg/liter of aprotinin(3-58, 42 Ser).

N-terminal sequencing carried out as described above confirmed the following N-terminal sequence ##STR8## i.e., the correct sequence.

The amino acid analysis is shown in Table 1 and confirms the expected amino acid composition, i.e., less pro and Arg and more Ser (see also the above remarks in Example 1).

When compared by the above mentioned method of Erlanger et al. the specific activity of aprotinin(3-58,42 Ser) was found to be identical within the experimental error with the specific activity of native aprotinin.

7.3. EXAMPLE 3: PRODUCTION OF APROTININ(1-58)

The synthetic duplex (SEQ ID NOS: 36 and 37) shown in FIG. 4 was ligated to the 330 bp EcoRI-HgaI fragment from plasmid pKFN9 coding for MFαl signal and leader sequence and to the 144 bp AvaII-XbaI fragment from pKFN305 and to the large EcoRI-XbaI fragment from pUC19.

The ligation mixture was used to transform a competent E. coli strain (r⁻ m⁺) selecting for ampicillin resistance. Sequencing of a ³² P-labelled XbaI-EcoRI fragment showed that plasmids from the resulting colonies contained the correct DNA sequence for aprotinin(1-58).

One plasmid pKFN414 was selected for further use. The construction of plasmid pKFN414 is illustrated in FIG. 4.

By following the procedure of Example 1 (Section 7.1., supra) a yeast plasmid pKFN418 was obtained containing the following construction:

    TPI.sub.P -MFαl-signal-leader(1-85)-aprotinin(1-58)-TPI.sub.T

Yeast strain MT633 was transformed with plasmid pKFN418 as described above. Culturing of the transformed strain KFN385 gave about 1-13 mg/l of aprotinin(1-58).

When compared with the above mentioned method of Erlanger et al., the specific activity of aprotinin(1-58), produced according to this example was found to be identical within the experimental error with the specific activity of native aprotinin. The sequence of the synthetic gene encoding aprotinin(1-58) is shown in FIG. 16 and set forth in the Sequence Listing as SEQ ID NOS: 38 and 39.

7.4 EXAMPLE 4: PRODUCTION OF APROTININ(1-58,42 Ser)

A plasmid pKFN416 containing a gene for aprotinin(1-58,42 Ser) was constructed from pKFN306 as described in Example 3 (Section 7.3., supra). The synthetic gene encoding aprotinin(1-58, 42 Ser) is shown in FIG. 17 and set forth in the Sequence Listing as SEQ ID NOS: 40 and 41. By following the procedure of Example 1 (Section 7.1., supra) a yeast plasmid pKFN420 was obtained containing the following construction:

    TPI.sub.P -MFαl-signal-leader(1-85)-aprotinin(1-58,42 Ser)-TPI.sub.T

Yeast strain MT663 was transformed with plasmid pKFN420 as described above. Culturing of the transformed strain KFN387 gave about 1-13 mg/l of aprotinin(1-58,42 Ser).

When compared with the above mentioned method of Erlanger et al., the specific activity of aprotinin(1-58,42 Ser) was found to be identical within the experimental error with the specific activity of native aprotinin.

7.5. EXAMPLE 5: APROTININ (3-58; 17 Ala+42 Ser) (KFN 396)

A sequence encoding aprotinin (3-58; 42 Ser) was constructed from a number of oligonucleotides by ligation using procedures described in Example 2 (Section 7.2., supra).

One plasmid, pKFN306, was selected for further use. The construction of plasmid pKFN306 is illustrated in FIG. 6.

To introduce Ala in position 17 the following oligonucleotides were synthesized as described below: ##STR9##

The oligonucleotides were 5'-phosphorylated by treatment with ATP and T4 kinase.

A duplex formed by annealing 5'-phosphorylated oligonucleotides Ia and IIa was ligated to the 352 bp EcoRI-Pf1MI fragment and the 3 kbp EcoRI-StyI fragment, both from pKFN306. pKFN306 encodes the S. cerevisiae mating factor αl signal leader (1-85) fused to the synthetic aprotinin (3-58; 42 Ser) gene.

The ligation mixture was used to transform a competent E. coli strain (r⁻, m⁺) selecting for ampicillin resistance. Sequencing of a ³² P-XbaI-EcoRI fragment (Maxam, A. and Gilbert, W. (1980) Methods Enzymol. 65: 499-560) showed that plasmids from the resulting colonies contained the correct DNA sequence for aprotinin (3-58; 17 Ala+42 Ser).

One plasmid, pKFN501 was selected for further use. The construction of plasmid pKFN501 is illustrated in FIG. 4.

pKFN501 was cut with EcoRI and XbaI and the 0.5 kb fragment was ligated to the 9.5 kb NcoI-XbaI fragment from pMT636 and the 1.4 kb NcoI-EcoRI fragment from pMT636, resulting in plasmid pKFN504 (see FIG. 4). Plasmid pMT 636 was constructed from pMT608 after deletion of the LEU-2 gene and from pMT479 (see FIG. 3). pMT608 is described in European Application No. 195691. pMT479 is described in European Patent Application NO. 163529. pMT479 contains the Schizo, pombe TPI gene (POT), the S. cerevisiae triosephosphate isomerase promoter and terminator, TPI_(P) and TPI_(T) (Alber, T. and Kawasaki, G. (1982) J. Mol. Appl. Gen. 1, 419-434). Plasmid pKFN504 contains the following sequence: TPI_(P) -MFαl-signal-leader(1-85)-aprotinin(3-58; 17 Ala+42 Ser)-TPI_(T) where MFαl is the S. cerevisiae mating factor alpha 1 coding sequence (Kurjan, J. and Herskowitz, I. (1982) Cell 30, 933-943), signal leader (1-85) means that the sequence contains the first 85 amino acid residues of the MFαl signal leader sequence and aprotinin (3-58; 17 Ala+42 Ser) is the synthetic sequence encoding an aprotinin derivative lacking the first two amino acid residues at the N-terminus and having amino acid residues 17 and 42 replaced by an Ala and a Ser residue, respectively.

S. cerevisiae strain MT663 (E2-7B XE11-36 a/α, ΔtpiΔtpi, pep 4-3/pep 4-3 ) was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an optical density at 600 nm of 0.6.

100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of (1.2M sorbitol, 25 mM Na₂ EDTA pH=8.0, 6.7 mg/ml dithiothreitol). The suspension was incubated at 30° C. for 15 minutes, centrifuged and the cells resuspended in 10 ml of (1.2M sorbitol, 10 mM Na₂ EDTA, 0.1M sodium citrate pH=5.8, 2 mg Novozym® 234). The suspension was incubated at 30° C. for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2M sorbitol and 10 ml of CAS (1.2M sorbitol, 10 mM CaCl₂, 10 mM Tris HCl (Tris=Tris (hydroxymethyl) aminomethane) pH=7.5) and resuspended in 2 ml of CAS. For transformation 0.1 ml of CAS resuspended cells were mixed with approximately 1 μg of plasmid pKFN504 and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4,000, 10 mM CaCl₂, 10 mM Tris HCl, pH=7.5) was added and the mixture left for further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2M sorbitol, 33% v/v YPD, 6.7 mM CaCl₂, 14 μg/ml leucine)) and incubated at 30° C. for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2M sorbitol. 6 ml of top agar (the SC medium of Sherman et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory, 1981) containing 1.2M sorbitol plus 2.5% agar) at 52° C. was added and the suspension poured on top of plates containing the same agar solidified, sorbitol containing medium. Transformant colonies were picked after 3 days at 30° C., reisolated and used to start liquid cultures. One such transformant KFN396 was chosen for further characterization.

Yeast strain KFN396 was grown on YPD medium (1% yeast extract, 2% peptone (from Difco Laboratories) and 2% glucose). A 1 liter culture of the strain was shaken at 30° C. to an optical density of 600 nm of 13. After centrifugation the supernatant was purified by FPLC ion exchange chromatography. The yeast supernatant was filtered through a 0.22 μm Millex® GV filter unit and 1 ml was applied on a MonoS cation exchange column (0.5×5 cm) equilibrated with 20 mM Bicine, pH 8.7. After wash with equilibration buffer the column was eluted with a linear NaCl gradient (0-1M) in equilibration buffer. Trypsin inhibitor activity was quantified in the eluted fractions by spectrophotometric assay and furthermore by integration of absorption at 280 nm from:

    E.sup.1%.sub.280 (aprotinin)=8.3

The yield was about 4.3 mg/liter of aprotinin (3-58; 17 Ala+42 Ser).

For amino acid analysis the yeast supernatant (7 ml) was adjusted to pH 8.7 with 0.1M NaOH and filtered (0.22 μm). The effluent from a Q-Sepharose anion exchange column (1×4 cm) equilibrated with 20 mM Bicine, pH 8.7 was applied to a MonoS cation exchange column (0.5×5 cm). The cation exchange chromatography was carried out as described above. Concentration of the gradient eluted aprotinin (3-58) was made by rechromatography on MonoS and elution with steep NaCl gradient. The collected fractions were further concentrated by vacuum centrifugation to about 100 μl and applied to a RP-HPLC column (Vydac C4, 4.6×250 mm). Elution was carried out with CH₃ CN gradient in 0.1% TFA. The collected fractions were concentrated to about 100 μl by vacuum centrifugation and samples were taken for amino acid analysis.

The amino acid analysis appears from the following Table 2. From this table it appears that the product has the expected amino acid composition:

    ______________________________________                                                              Aprotinin                                                 Amino                (3-58; 17 Ala + 42 Ser                                    Acid       Theoretical                                                                              (Found)                                                   ______________________________________                                         Asx        5         4.90                                                      Thr        3         2.95                                                      Ser        2         2.10                                                      Glx        3         3.01                                                      Pro        3         3.14                                                      Gly        6         5.93                                                      Ala        7         6.69                                                      Cys        6         5.91                                                      Val        1         1.02                                                      Met        1         0.99                                                      Ile        2         2.00                                                      Leu        2         1.98                                                      Tyr        4         3.73                                                      Phe        4         3.75                                                      Lys        4         4.29                                                      Arg        3         3.21                                                      Total      56        55.60                                                     ______________________________________                                    

7.6. EXAMPLE 6: Aprotinin (3-58; 17 Ala+19 Glu+42 Ser) (KFN 399)

A synthetic gene encoding aprotinin (3-58; 17 Ala+19 Glu+42 Ser) was constructed as described in Example 5 (Section 7.5., supra). The following oligonucleotides Ib and IIb were used instead of Ia and IIa: ##STR10##

The pUC19 derived plasmid pKFN503 was constructed in a similar way as pKFN501.

By following the procedure of Example 5 a plasmid PKFN507 was obtained containing the following construction: TPI_(P) -MFαl-signal-leader(1-85)-aprotinin(3-17 Ala+19 Glu+42 Ser)-TPI_(T), where aprotinin (3-58; Ala+19 Glu+42 Ser) is the synthetic gene encoding an aprotinin derivative lacking the first two amino acid residues at the N-terminal and having the residues 17, 19 and 42 of native aprotinin replaced by an alanine, a glutamic acid and a serine residue, respectively.

Plasmid pKFN507 was transformed in yeast strain MT663 as described above and culturing of the transformed strain KFN399 gave about 10 mg/liter of aprotinin (3-58; 17 Ala+19 Glu+42 Ser).

The amino acid analysis appears from the following Table 3 and confirms the expected amino acid composition:

    ______________________________________                                                          Aprotinin                                                     Amino            (3-58; 17 Ala + 19 Glu + 42 Ser)                              Acid   Theoretical                                                                              (Found)                                                       ______________________________________                                         Asx    5         4.95                                                          Thr    3         2.83                                                          Ser    2         1.90                                                          Glx    4         4.08                                                          Pro    3         2.98                                                          Gly    6         5.98                                                          Ala    7         6.92                                                          Cys    6         5.06                                                          Val    1         0.99                                                          Met    1         0.86                                                          Ile    1         0.99                                                          Leu    2         1.99                                                          Tyr    4         3.77                                                          Phe    4         3.89                                                          Lys    4         4.07                                                          Arg    3         3.06                                                          Total  56        54.36                                                         ______________________________________                                    

7.7 EXAMPLE 7: APROTININ (3-58; 15 Arg+17 Ala+42 Ser) (KFN 773)

A synthetic gene encoding aprotinin (3-58; 15 Arg+17 Ala+42 Ser) was constructed as described in Example 5 (See Section 7.5., supra). The following oligonucleotides Ic and IIc were used instead of Ia and IIa: ##STR11##

The pUC19 derived plasmid pKFN777 was constructed in a similar way as pKFN501.

By following the procedure of Example 5 (Section 7.5, supra) a plasmid pKFN807 was obtained containing the following construction: TPI_(P) -MFαl-signal-leader(1-85)aprotinin (3-58; 15Arg+17Ala+42Ser)-TPI_(T), where aprotinin (3-58; 15 Arg+17 Ala+42 Ser) is the synthetic gene encoding an aprotinin derivative lacking the first two amino acid residues at the N-terminal and having the residues 15, 17 and 42 of native aprotinin replaced by an arginine, an alanine and a serine residue, respectively.

Plasmid DKFN807 was transformed in yeast strain MT663 as described above and culturing of the transformed strain KFN773 gave about 8.5 mg/liter of aprotinin (3-58; 15 Arg+17 Ala+42 Ser).

The amino acid analysis is shown in Table 4 and confirms the expected amino acid composition:

    ______________________________________                                                            Aprotinin                                                   Amino              (3-58; 17 Arg + 17 Ala + 42 Ser)                            Acid     Theoretical                                                                              (Found)                                                     ______________________________________                                         Asx      5         4.95                                                        Thr      3         2.85                                                        Ser      2         1.81                                                        Glx      3         3.01                                                        Pro      3         3.05                                                        Gly      6         5.92                                                        Ala      7         6.91                                                        Cys      6         5.31                                                        Val      1         1.02                                                        Met      1         0.73                                                        Ile      2         1.41                                                        Leu      2         1.99                                                        Tyr      4         3.80                                                        Phe      4         3.94                                                        Lys      3         2.97                                                        Arg      4         4.24                                                        Total    56        53.91                                                       ______________________________________                                    

The slightly lowered content of Ile compared with the theoretical value can most probably be ascribed to incomplete hydrolysis of Ile (18)-Ile (19). This is well known in the art.

7.8. EXAMPLE 8: INHIBITION OF SERINE PROTEASES FROM PLASMA BY APROTININ (3-58; 17 Ala+42 Ser) (KFN 396) AND APROTININ (3-58; 17 Ala+19 Glu+42 Ser) (KFN 399), APROTININ (3-58; 15 Arg+42 Ser) (KFN 772) AND APROTININ (3-58; 15 Arg+17 Ala+42 Ser) (KFN 773).

Aprotinin (3-58; 17 Ala+42 Ser; SEQ ID NO: 5) (KFN 396), aprotinin (3-58; 17 Ala+19 Glu+42 Ser; SEQ ID NO: 6) (KFN 399) and aprotinin (3-58; 15 Arg+17 Ala+42 Ser; SEQ ID NO: 7) (KFN 773) were purified as described above. As native, bovine pancreatic aprotinin (1-58; SEQ ID NO: 1) batch B 5029-65 (67,000 KIU/mg) from NOVO (Bagsvaerd, Denmark) was used. The concentration was calculated using E₂₈₀ nm=8.3 and M_(r) =6,500. Human plasma kallikrein was obtained from Sigma (St. Louis, Mo.), bovine factor Xa was purified according to (H. Nobukazu et al. J. Biochem. 97 (1985) 1347-1355), human factor IIa (thrombin) was a gift from Dr. W. Lawson (New York State Department of Health, Albany, N.Y.), recombinant human factor VIIa was from NOVO (Bagsvaerd, Denmark) and recombinant human protein Ca was from ZymoGenetics, Inc. (Seattle, Wash). Substrate S 2302 (H-D-Pro-Phe-Arg-p-nitroanilide) substrate S2238 (H-D-Phe-Pip-Arg-p-nitroanilide) and substrate S2366 (Glu-Pro-Arg-p-nitroanilide) were from Kabi (Stockholm, Sweden). Substrate FXa-1 (methoxycarbonyl DCH-Gly-Arg-p-nitroanilide) was from NycoMed (Oslo, Norway). The experiments were performed in 100 mM NaCl, 50 mM Tris-HCl 0.01% Tween80, pH 7.4 at 25° C.

Human plasma kallikrein (3 nM) was incubated with aprotinin (0-20 nM) for 30 minutes in a micro-titer well. Substrate S 2302 (0.6 mM) was added to a final volume of 300 μl and the rate of nitroaniline generation was measured at 405 nm by means of a Micro ELISA® Autoreader MR 580 from Dynatech Laboratories. The rate is proportional to the concentration of free enzyme. The inhibition of plasma kallikrein by native aprotinin and the 4 analogues KFN 396, KFN 399, KFN 772 and KFN 773 is shown in FIGS. 9A and 9B. With native aprotinin a moderate inhibition was observed. The inhibition was strongly increased by analogs KFN 396 and KFN 399 containing Ala in position 17 (FIG. 9A).

A further increase of the inhibition was obtained with Arg in position 15 (KFN 772); and the strongest inhibition was observed with the analog (KFN 773) with substitution of both position 17 (Ala) and position 15 (Arg) (FIG. 9B).

The analogs were also tested for inhibition of the amidolytic activity of the serine proteases: bovine factor Xa, human factor IIa, human recombinant factor VIIa and human recombinant protein Ca. The experiments were performed essentially as described for plasma kallikrein only appropriate substrates were used. Finally the analogs were analyzed for an effect on the coagulation factors of human plasma by means of two clotting tests. These tests, the prothrombin time (PTT) and the activated thromboplastin time (ATPT) were performed with General Diagnostics® reagents from Organon (Durham, N.C.) according to the directions given by the manufacturer. The results of the inhibition experiments are summarized in Table 5 which describes the inhibition profile of the 4 aprotinin analogs. KFN 773 is characterized by an extraordinarily strong inhibition of human plasma kallikrein which is ten-fold stronger than that of the Arg 15 analogue (KFN 772). A reverse effect is observed with activated protein C. In this case, the relatively strong inhibition obtained by substitution of Lys 15 to Arg is weakened by further substitution of Arg 17 to Ala.

                                      TABLE 5                                      __________________________________________________________________________           K.sub.i *.sup.) (nM); Amidolytic                                               Activity                                                                       of Serine Proteases §                                                                               Clot                                                 Plasma                 Prot.                                                                             Assays                                         Product                                                                              Kallikrein FIIa                                                                               FVIIa                                                                              FXa Ca PTT  APTT                                      __________________________________________________________________________     Native                                                                               180        --  --  --  400                                                                               --   --                                        Aprotinin                                                                      KFN 396                                                                              12         --  --  --     --   --                                        KFN 399                                                                              12                                                                       KFN 772                                                                               1         --  --  1,800                                                                               10                                                                               --   +                                         KFN 773                                                                              0.1        --  --    150                                                                              100                                                                               --   +                                         __________________________________________________________________________      -- No inhibition at 1.0 μM aprotinin analog                                 + Prolonged clotting time at 1.0 μM aprotinin analog                        *.sup.) inhibition constants estimated according to the graphical Dixon        method (M. Dixon, Biochem. J. 129 (1972) 197-202)                              § Substrates: Plasma kallikrein: S2302; FIIa: S2238; FVIIa: Substrat      FXa1; FXa: Substrate FXa1; Prot. Ca: S2366.                              

7.9. EXAMPLE 9: PRODUCTION OF [Glu1, Glu26, Glu41, Glu46]-APROTININ FROM YEAST STRAIN KFN-1512

A synthetic gene coding for [Glu1, Glu26, Glu41, Glu46]-aprotinin was constructed from 10 oligonucleotides by ligation.

The oligonucleotides were synthesized on an automatic DNA synthesizer using phosphoramidite chemistry on a controlled pore glass support (Beaucage, S. L., and Caruthers, M. H., Tetrahedron Letters 22, (1981) 1859-1869).

The following 10 oligonucleotides were synthesized: ##STR12##

5 duplexes A-E were formed from the above 10 oligonucleotides as shown in FIG. 10. 20 pmole of each of the duplexes A-E were formed from the corresponding pairs of 5'-phosphorylated oligonucleotides by heating for 5 min. at 90° C. followed by cooling to room temperature over a period of 75 minutes. The five duplexes were mixed and treated with T₄ DNA ligase. The synthetic gene was isolated as a 203 bp band after electrophoresis of the ligation mixture on a 2% agarose gel. The obtained synthetic gene is shown in FIG. 10 and is set forth as SEQ ID NO: 28. The synthetic gene was ligated to a 209 bp EcoRI-NcoI fragment from pLaC212spx3 and to the 2.8 Kb EcoRI-XbaI fragment of plasmid pTZ19R (Mead, D. A., Szczesna-Skorupa, E. and Kemper, B., Prot. Engin. 1 (1986) 67-74). Plasmid pLaC212spx3 is described in Example 3 of International Patent Application No. PCT/DK88/00147. The 209 bp EcoRI-NcoI fragment from pLaC212spx3 encodes a synthetic yeast leader peptide.

The ligation mixture was used to transform a competent E. coli strain r⁻, m⁺) selecting for ampicillin resistance. DNA sequencing (Sanger, F., Micklen, S., and Coulson, A. R., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467) showed that plasmids from the resulting colonies contained the correct DNA sequence for [Glu1, Glu26, Glu41, Glu46]-aprotinin.

One plasmid pKFN-1503 was selected for further use. The construction of plasmid DKFN-1503 is illustrated in FIG. 11. pKFN-1503 was cut with EcoRI and XbaI and the 412 bp fragment was ligated to the 9.5 kb NcoI-XbaI fragment from pMT636 and the 1.4 kb NcoI-EcoRI fragment from pMT636, resulting in plasmid DKFN-1508 (see FIG. 11). Plasmid pMT636 is described in International Patent Application No. PCT/DK88/00138.

pMT636 is an E. coli--S. cerevisiae shuttle vector containing the Schizosaccharomyces pombe TPI gene (POT) (Russell, P. R., Gene 40 (1985) 125-130), the S. cerevisiae triosephosphate isomerase promoter and terminator, TPI_(P) and TPI_(T) (Alber, T., and Kawasaki, G. J. Mol. Appl. Gen. 1 (1982), 419-434). Plasmid pKFN-1508 contains the following sequence: TPI_(P) -LaC212spx3 signal-leader (1-47)-Glu(ArgLeuGluLysArg [Glu1, Glu26, Glu41, Glu46]-aprotinin-TPI_(T) where LaC212spx3 signal-leader is the synthetic yeast leader described in International Patent Application No. PCT/DK88/00147. The DNA sequence of the 412 bp EcoRI-XbaI fragment from pKFN-1503 and pKFN-1508 is SEQ ID NOS: 52 and 53.

S. cerevisiae strain MT663 (E2-7B XE11-36 a/α, tpi/ tpi, pep 4-3/pep 4-3) was grown on YPGaL (1% Bacto yeast extract, 2% Bacto peptone, 2% galactose, 1% lactate) to an O.D. at 600 run of 0.6.

100 ml of culture was harvested by centrifugation, washed with 10 ml of water, recentrifuged and resuspended in 10 ml of a solution containing 1.2M sorbitol, 25 mM Na₂ EDTA pH=8.0 and 6.7 mg/ml dithiothreitol. The suspension was incubated at 30° C. for 15 minutes, centrifuged, and the cells resuspended in 10 ml of a solution containing 1.2M sorbitol, 10 mM Na₂ EDTA, 0.1M sodium citrate, pH=5.8, and 2 mg Novozym® 234. The suspension was incubated at 30° C. for 30 minutes, the cells collected by centrifugation, washed in 10 ml of 1.2M sorbitol and 10 ml of CAS (1.2M sorbitol, 10 mM CaCl₂, 10 mM Tris HCl (Tris=Tris(hydroxymethyl)-aminomethane, pH=7.5) and resuspended in 2 ml of CAS. For transformation, 0.1 ml of CAS-resuspended cells were mixed with approx. 1 μg of plasmid pKFN-1508 and left at room temperature for 15 minutes. 1 ml of (20% polyethylene glycol 4000, 20 mM CaCl₂, 10 mM CaCl₂, 10 mM Tris HCl pH=7.5) was added and the mixture left for a further 30 minutes at room temperature. The mixture was centrifuged and the pellet resuspended in 0.1 ml of SOS (1.2M sorbitol, 33% v/v YPD, 6.7 mM CaCl₂, 14 μg/ml leucine) and incubated at 30° C. for 2 hours. The suspension was then centrifuged and the pellet resuspended in 0.5 ml of 1.2M sorbitol. Then, 6 ml of top agar (the SC medium of Sherman et al., (Methods in Yeast Genetics, Cold Spring Harbor Laboratory (1982)) containing 1.2M sorbitol plus 2.5% agar) at 52° C. was added and the suspension poured on top of plates containing the same agar-solidified, sorbitol containing medium.

Transformant colonies were picked after 3 days at 30° C., reisolated and used to start liquid cultures. One such transformant KFN-1512 was selected for further characterization.

Yeast strain KFN-1512 was grown on YPD medium (1% yeast extract, 2% peptone (from Difco Laboratories), and 6% glucose). A 200 ml culture of the strain was shaken at 250 rpm at 30° C. for 3 days to an O.D. at 600 nm of about 20. After centrifugation, the supernatant was analyzed by FPLC ion exchange chromatography. The yeast supernatant was filtered through a 0.22 μm Millex GV filter unit, and 1 ml was applied on a MonoS cation exchange column (0.5×5 cm) equilibrated with 20 mM formic acid, pH 3.7. After washing with equilibration buffer, the column was eluted with a linear NaCl gradient (0.1M) in equilibration buffer. Trypsin inhibitor activity was quantified in the eluted fractions by spectrophotometric assay (Kassel, B., Methods Enzymol. 19 (1970), 844-852) and furthermore by integration of absorption at 280 nm from

    E.sup.1%.sub.280 (aprotinin)=8.3

In order to obtain material for toxicology studies, yeast strain KFN-1512 was grown on a larger scale. The aprotinin analog was purified by a combination of ion exchange chromatography and reverse phase HPLC.

7.10. EXAMPLE 10: PRODUCTION OF [Glu1, Glu42, Glu46]APROTININ FROM YEAST STRAIN KFN-1514

A synthetic gene coding for [Glu1, Glu42, Glu46]-aprotinin was constructed from 10 oligonucleotides by ligation as described in Example 9.

The pTZ19R-derived plasmid pKFN-1505 containing the synthetic gene fused in frame to a synthetic yeast leader peptide was constructed as described in Example 9.

By following the procedure of Example 1, a yeast expression plasmid pKFN-1510 was obtained containing the following construction TPI_(P) -LaC212spx3 signal- leader (1-47)-GluArgLeuGluLysArg [Glu1, Glu42, Glu46]-aprotinin-TPI_(T).

The DNA sequence of the 412 bp EcoRI-XbaI fragment from DKFN-1505 and pKFN-1510 is SEQ ID NOS: 54 and 55. Plasmid pKFN-1510 was transformed in yeast strain MT663 as described a/Dove resulting in yeast strain KFN-1514.

Culturing of the transformed strain KFN-1514 in YPD-medium, analysis for [Glu1, Glu42, Glu46]-aprotinin in the supernatant, and production of material for toxicological studies was performed as described above.

7.11. EXAMPLE 11: PRODUCTION OF [Glu42, Glu46]-APROTININ FROM YEAST STRAIN KFN-1544

The 144 bp AvaII-XbaI fragment encoding [Glu42, Glu46]-aprotinin (12-58) from pKFN-1505 was used to replace the corresponding DNA fragment encoding aprotinin(12-58) from plasmid pKFN-1000 resulting in plasmid pKFN-1528. Plasmid pKFN-1000 is described in Example 4 of International Patent Application, Publication No. WO 90/10075.

By following the procedure of Example 9, a yeast expression plasmid pKFN-1541 was obtained containing the following construction TPI_(P) -LaC212spx3signal-leader (1-47)-GluArgLeuGluLysArg-[Glu42, Glu46]-aprotinin-TPI_(T).

The DNA sequence of the 412 bp EcoRI-XbaI fragment from pKFN-1528 and pKFN-1541 is SEQ ID NOS: 56 and 57. Plasmid pKFN-1541 was transformed in yeast strain MT663 as described above resulting in yeast strain pKFN-1544.

Culturing of the transformed strain KFN-1544 in YPD-medium, analysis for [Glu42, Glu46]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.12. EXAMPLE 12: PRODUCTION OF [Ser10, Asp24, Thr26, Glu31, Asn41, Glu53]-APROTININ FROM YEAST STRAIN KFN-15-45

The synthetic gene coding for [Ser10, Asp24, Thr26, Glu31, Asn41, Glu53] was constructed from 10 oligonucleotides by ligation as described in Example 9 (see Section 7.9., supra).

The pTZ19R-derived plasmid pKFN-1530 containing the synthetic gene fused in frame to a synthetic yeast leader peptide sequence was constructed as described in Example 9 (see Section 7.9., supra).

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1532 was obtained containing the following construction TPI_(P) -LaC212spx3 signal-leader(1-47)-GluArgLeuGluLysArg-[Ser10, Asp24, Thr26, Glu31, Asn41, Glu52]-aprotinin-TPI_(T). The DNA sequence of the 412 bp EcoRI-XbaI fragment from pKFN-1530 and pKFN-1532 is SEQ ID NOS: 58 and 59.

Plasmid pKFN-1532 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1545.

Culturing of the transformed strain KFN-1545 in YPD-medium, analysis for [Ser10, Asp24, Thr26, Glu31, Asn41, Glu53]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.13. EXAMPLE 5: PRODUCTION OF [Ser10, Leu20, Gly40, Asn41, Gln44, Tyr46]-APROTININ FROM YEAST STRAIN KFN-1547

The synthetic gene coding for [Ser10, Leu20, Gly40, Asn41, Gly42, Gln44, Tyr46]-aprotinin was constructed from 10 oligonucleotides by ligation as described in Example 9 (see Section 7.9., supra).

The pTZ19R-derived plasmid pKFN-1534 containing the synthetic gene fused in frame to a synthetic yeast leader peptide sequence was constructed as described in Example 9 (see Section 7.9., supra).

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1537 was obtained containing the following construction TPI_(P) -LaC212spx3 signal-leader(1-47)-GluArgLeuGluLysArg-[Ser10, Leu20, Gly40, Asn41, Gly42, Gln44, Tyr46]-aprotinin-TPI_(T). The DNA sequence of the 412 bp EcoRI-XbaI fragment from pKFN-1534 and pKFN-1537 is SEQ ID NOS: 60 and 61. Plasmid pKFN-1537 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1547.

Culturing of the transformed strain KFN-1547 in YPD-medium, analysis for [Ser10, Leu20, Gly40, Asn41, Gly42, Gln44, Tyr46]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.14. EXAMPLE 14: PRODUCTION OF Des-Argl, des-Pro2-[Ser 42,Glu46]-APROTININ FROM YEAST STRAIN KFN-1660

The 1.4 kbAhaII-StyI fragment and the 1.8 kb AhaII-SalI fragment both from plasmid pKFN-306 were ligated to a duplex consisting of the following two synthetic oligonucleotides: ##STR13##

Plasmid pKFN-306 is a pTZ19R derived plasmid with a 502 bp EcoRI-XbaI insert containing the Saccharomyces cerevisiae mating factor alpha-1-signal-leader (1-85) gene fused in-free with a synthetic gene for des-Argl, des-Pro2-[Ser42]-aprotinin. The construction of plasmid pKFN-306 is described in WO 89/01968.

The ligation mixture was used to transform a competent E. coli strain (r⁻, m⁺) selecting for ampicillin resistance. DNA sequencing (Sanger, F., Micklen, S., and Coulsen, A. R., Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467) showed that plasmids from the resulting colonies contained the correct sequence for des-Argl, Pro2-[Ser42, Glu46] aprotinin.

One plasmid pKFN-1629 was selected for further use.

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1656 was obtained containing the following construction: TPI_(P) -MFαl signal-leader(1-85)-des-Argl, des-Pro2-[Ser42, Glu46]-aprotinin-TPI_(T).

The DNA sequence of the 502 bp EcoRI-XbaI fragment from pKFN-1629 and pKFN-1656 is SEQ ID NOS: 64 and 65.

Plasmid pKFN-1656 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1660.

Culturing of the transformed strain KFN-1660 in YPD-medium, analysis for des-Argl, des-Pro2-[Ser42, Glu46]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.15. EXAMPLE 15: PRODUCTION OF Des-Argl, des-Pro2-[Ser42,Ala46]-APROTININ FROM YEAST STRAIN KFN-1661

The 1.4 kbAhaII-StyI fragment and the 1.8 kb AhaII-SalI fragment both from plasmid pKFN-306 were ligated to a duplex consisting of the following two synthetic oligonucleotides: ##STR14##

Plasmid pKFN-306 is a pTZ19R derived plasmid with a 502 bp EcoRI-XbaI insert containing the Saccharomyces cerevisiae mating factor alpha 1 signal-leader (1-85) gene fused in-frame with a synthetic gene for des-Argl, des-Pro2-[Ser42]-aprotinin. The construction of plasmid pKFN-306 is described in Example 3 (see Section 7.3., supra).

The ligation mixture was used to transform a competent E. coli strain (r⁻, m⁺) selecting for ampicillin resistance. DNA sequencing (Sanger, F., Micklen, S., and Coulsen, A. R., Proc. Natl. Acad. Sci. USA74 (1977) 5463-5467) showed that plasmids from the resulting colonies contained the correct sequence for des-Argl, des-Pro2-[Ser42,Glu46] aprotinin. One plasmid pKFN-1631 was selected for further use.

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1657 was obtained containing the following construction: TPI_(P) -MFαl signal-leader(1-85)-des-Argl, des-Pro2-[Ser42,Ala46]-aprotinin-TPI_(T).

The DNA sequence of the 502 bp EcoRI-XbaI fragment form pKFN-1631 and pKFN-1657 is SEQ ID NOS: 68 and 69.

Plasmid pKFN-1657 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1661.

Culturing of the transformed strain KFN-1661 in YPD-medium, analysis for des-Argl, des-Pro2-[Ser42,Ala46]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.16. EXAMPLE 16: PRODUCTION OF Des-Argl,des-Pro2-[Ser10, Asp24, Thr26, Glu31, Asn41, Glu53]-APROTININ FROM YEAST STRAIN KFN-1735

A synthetic gene coding for des-Argl,des-Pro2-[Ser10, Asp24, Thr26, Glu31, Asn41, Glu53]-aprotinin was constructed from 10 oligonucleotides by ligation as described in Example 9 (see Section 7.9., supra).

The pTZ19R-derived plasmid pKFN-1707 containing the synthetic gene fused in frame to a synthetic yeast leader peptide was constructed as described in Example 9 (see Section 7.9., supra).

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1709 was obtained containing the following construction TPI_(P) -LaC212spx3signal-leader(1-47) -GluArgLeuGluLysArg-des Argl,des-Pro2-[Ser10, Asp24, Thr26, Glu31, Asn41, Glu53]-aprotinin-TPI_(T).

The DNA sequence of the 406 bp EcoRI-XbaI fragment from pKFN-1707 and pKFN-1709 is SEQ ID NOS: 70 and 71.

Plasmid pKFN-1709 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1735.

Culturing of the transformed strain KFN-1735 in YPD-medium, analysis for des-Argl, des-Pro2-[Ser10, Asp24, Thr26, Glu31, Asn41, Glu53]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.17. EXAMPLE 17: PRODUCTION Of Des-Argl,des-Pro2-[Asp24, Thr26, Glu31, Glu53]-APROTININ FROM YEAST STRAIN KFN-1737

The 1.8 kb AhaII-XbaI fragment and the 1.4 kb AhaII-AvaII fragment both from plasmid pKFN-306 (see example 5) were ligated to a synthetic 141 bp AvaII-XbaI fragment encoding [Asp24, Thr26, Glu31, Glu53]-aprotinin. The resulting pTZ19R-derived plasmid was pKFN-1711.

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1713 was obtained containing the following construction TPI_(P) -MFαl signal-leader (1-85)-des-Argl, des-Pro2-[Asp24, Thr26, Glu31, Glu53]-aprotinin-TPI_(T).

The DNA sequence of the 502 bp EcoRI-XbaI fragment from pKFN-1711 and pKFN-1713 is SEQ ID NOS: 72 and 73.

Plasmid pKFN-1713 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1737. Culturing of the transformed strain KFN-1737 in YPD-medium, analysis for des-Argl, des-Pro2-[Asp24, Thr26, Glu31, Glu53]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.18. EXAMPLE 18: PRODUCTION Of Des-Argl, des-Pro2-[Ser10, Gly40, Asn41, Gly42, Glu53]-APROTININ FROM YEAST STRAIN KFN-1739

A synthetic gene coding for des-Argl,des-Pro2-[Ser10, Gly40, Asn41, Gly42, Glu53]-aprotinin was constructed from 10 oligonucleotides by ligation as described in Example 9 (see Section 7.9., supra).

The pTZ19R-derived plasmid pKFN-1715 containing the synthetic gene fused in frame to a synthetic yeast leader peptide was constructed as described in Example 9 (see Section 7.9., supra).

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1718 was obtained containing the following construction TPI_(P) -LaC212spx3 signal-leader(1-47)-GluArgLeuGluLysArg-des Argl,des-Pro2-[Ser10, Gly40, Asn41, Gly42, Glu53]-aprotinin-TPI_(T).

The DNA sequence of the 406 bp EcoRI-XbaI fragment from pKFN-1715 and pKFN-1718 is SEQ ID NOS: 74 and 75.

Plasmid pKFN-1718 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1739. Culturing of the transformed strain KFN-1739 in YPD-medium, analysis for des-Argl, Pro2-[Ser10, Gly40, Asn41, Gly42, Glu53]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.19. EXAMPLE 19: PRODUCTION OF Des-Argl,des-Pro2-[Ser42, Glu53]-APROTININ FROM YEAST STRAIN KFN-1742

The 1.8 kb AhaII-XbaI fragment and the 1.4 kb AhaII-AvaII fragment both from plasmid pKFN-306 (see example 5) were ligated to a synthetic 141 bp AvaII-XbaI fragment encoding [Ser42, Glu53]-aprotinin. The resulting pTZ19R-derived plasmid was pKFN-1721.

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1724 was obtained containing the following construction TPI_(P) -MFαl signal-leader(1-85)-des-Argl, des-Pro2-[Ser42, Glu53]-aprotinin-TPI_(T).

The DNA sequence of the 502 bp EcoRI-X2DaI fragment from pKFN-1721 and pKFN-1724 is SEQ ID NOS: 76 and 77.

Plasmid pKFN-1724 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1742. Culturing of the transformed strain KFN-1742 in YPD-medium, analysis for des-Argl, des-Pro2-[Ser42, Glu53]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.20. EXAMPLE 20: PRODUCTION OF Des-Argl, des-Pro2-[Glu42, Glu53]-APROTININ FROM YEAST STRAIN KFN-1752

The 1.8 kb AhaII-XbaI fragment and the 1.4 kb AhaII-AvaII fragment both from plasmid pKFN-306 (see Example 14) were ligated to a synthetic 141 bp AvaII-XbaI fragment encoding [Glu42, Glu53]-aprotinin. The resulting pTZ19R- derived plasmid was pKFN-1762.

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1765 was obtained containing the following construction TPI_(P) -MFαl signal-leader(1-85)-des-Argl, des-Pro2-[Glu42, Glu53]-aprotinin-TPI_(T).

The DNA sequence of the 502 bp EcoRI-XbaI fragment from pKFN-1762 and pKFN-1765 is SEQ ID NOS: 78 and 79.

Plasmid pKFN-1765 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1752. Culturing of the transformed strain KFN-1752 in YPD-medium, analysis for des-Argl,des-pro2-[Glu42, Glu53]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.21. EXAMPLE 21: PRODUCTION OF Des-Argl,des-Pro2-[Glu26, Ser42, Glu53]-APROTININ FROM YEAST STRAIN KFN-1755

The 1.8 kb AhaII-XbaI fragment and the 1.4 kb AhaII-AvaII fragment both from plasmid pKFN-306 (see Example 14) were ligated to a synthetic 141 bp AvaII-XbaI fragment encoding [Glu26, Ser42, Glu53]-aprotinin. The resulting pTZ19R-derived plasmid was pKFN-1768.

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1770 was obtained containing the following construction TPI_(P) -MFαl signal-leader(1-85)-des-Argl,des-Pro2-[Glu26, Ser42, Glu53]-aprotinin-TPI_(T).

The DNA sequence of the 502 bp EcoRI-XbaI fragment from pKFN-1768 and pKFN-1770 is SEQ ID NOSS: 80 and 81 and 65.

Plasmid pKFN-1770 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1755. Culturing of the transformed strain KFN-1755 in YPD-medium, analysis for des-Argl,des-Pro2-[Glu26, Ser42, Glu53]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.22. EXAMPLE 22: PRODUCTION OF Des-Argl,des-Pro2-[Glu26, Glu42, Glu53]-APROTININ FROM YEAST STRAIN KFN-1756

The 1.8 kb AhaII-XbaI fragment and the 1.4 kb AhaII-AvaII fragment both from plasmid pKFN-306 (see Example 5) were ligated to a synthetic 141 bp AvaII-XbaI fragment encoding [Glu26, Glu42, Glu53]-aprotinin. The resulting pTZ19R-derived plasmid was pKFN-1771.

By following the procedure of Example 9 (see Section 7.9., supra), a yeast expression plasmid pKFN-1773 was obtained containing the following construction: TPI_(P) -MFαl signal-leader(1-85)-des-Argl,des-Pro2-[Glu26, Glu42, Glu53]-aprotinin-TPI_(T).

The DNA sequence of the 502 bp EcoRI-XbaI fragment from pKFN-1771 and pKFN-1773 is SEQ ID NOS: 82 and 83.

Plasmid pKFN-1773 was transformed in yeast strain MT663 as described above resulting in yeast strain KFN-1756. Culturing of the transformed strain KFN-1756 in YPD-medium, analysis for des-Argl,des-Pro2-[Ser42, Glu42, Glu53]-aprotinin in the supernatant and production of material for toxicological studies was performed as described above.

7.23. EXAMPLE 23: TOXICOLOGICAL SCREENING OF APROTININ ANALOGS BY SINGLE-DOSE INTRAVENOUS ADMINISTRATION TO WISTAR RATS 7.23.1. MATERIALS

The following aprotinin analogs with a reduced positive net charge and thermal stability compared to recombinant aprotinin (1-58) were selected for toxicological screening: KFN-1512, KFN-1514, KFN-1544, KFN-1545, KFN-1547, KFN-1660, KFN-1661, KFN-322, KFN-324, KFN-396, KFN-399, KFN-430, and KFN-773. Their main characteristics as seen from a toxicological point of view, are shown in Table 6. Data for recombinant aprotinin are shown for comparison. The denaturation temperature is shown as an indication of biological stability.

                  TABLE 6                                                          ______________________________________                                         General Information                                                                                            Denaturation                                   KFN-Type Chain Length Net Charge                                                                               T, °C.                                  ______________________________________                                         rAprotinin                                                                              1-58         +6        >100                                           1512     1-58         -2        87                                             1514     1-58          0        88                                             1544     1-58         +2        98                                             1545     1-58          0        93                                             1547     1-58         +2        86                                             1660     3-58         +2        77                                             1661     3-58         +3        79                                             1735     3-58         -1        68                                             1737     3-58          0        70                                             1739     3-58         +1        81                                             1742     3-58         +2        71                                             1752     3-58         +1                                                       1755     3-58          0        68                                             1756     3-58         -1        70                                              322     3-58         +5        84                                              324     3-58         +4        84                                              396     3-58         +3        75                                              399     3-58         +2        67                                              430     3-58         +3        70                                              773     3-58         +3        71                                             ______________________________________                                    

7.23.2. DESIGN

On Day 1 of the screening of each analog, groups of 2 male and 2 female rats received 33, 100, 300, or 900 mg analog/kg body weight. Two similarly constituted control groups received physiological saline or physiological saline acidified with hydrochloric acid to an approximate pH 4.5. The latter solution served as vehicle. The dose volume was 10 ml/kg body weight in all cases. The rats were observed for 7 days and killed on Day 8. At autopsy the kidneys were weighed and prepared for histopathology. Response variables are shown in the heading of Table 7.

7.23.3. RESULTS

Results from individual screenings are summarized in Table 7. Data for recombinant aprotinin are included for comparison (dosage: 11-300 mg/kg). KFN-1512 could not be dissolved as required for administration of the top dose (900 mg/kg).

A single animal died at 900 mg/kg KFN-1545 and all animals receiving a dose of 900 mg/kg KFN-322, KFN-430, and 773 were found dead/killed in extremis by Day 1-2. Apart from this, no fatalities were seen.

No histopathological kidney change was seen after administration 900 mg/kg of KFN-399, or after 300 mg/kg body weight of KFN-1512, KFN-1544, KFN-1545, KFN-396, KFN-430, KFN-773, and KFN-1660, or after 100 mg/kg of KFN-322 and KFN-324. Furthermore, no histopathological kidney change was seen after administration of 900 mg/kg body weight of KFN-1514, KFN-1547, and KFN-1661. Thus all analogs had no-toxic-effect levels concerning histopathological kidney change of 300 mg/kg or above, as compared to 11 mg/kg for aprotinin.

With respect to the other response variables the analogs equaled or were superior to aprotinin.

                                      TABLE 7                                      __________________________________________________________________________     No-toxic-effect levels by response variable, mg/kg                                    Clinical Observations                                                          0-30 min                                                                            2 hours                    Body                                                                               Kidney                                     after                                                                               after        Macroscopic                                                                           Microscopic                                                                           weight                                                                             weight                              KFN-type                                                                              dosage                                                                              dosage                                                                             Daily                                                                              Mortality                                                                           observations                                                                          observations                                                                          Day 8                                                                              Day 8                               __________________________________________________________________________     rAproti-                                                                               33  300 300 300   33     11    100 100                                 nin.sup.1,2                                                                    .sup. 1512.sup.1                                                                       33  300 300 300  300    300    300 300                                 1514   900  900 900 900  900    900    900 900                                 1544    33  100 100 900  300    300    900 300                                 1545    33  900 300 300  300    300    300 300                                 1547    33  300 900 900  900    900    900 900                                 1660   300  900 900 900  900    900    900 900                                 1661   100  900 900 900  300    900    900 900                                  322   100  900 300 300  300    100    100 300                                  324    33  300 300 900  300    100    300 300                                  396   300  900 900 900  900    300    300 900                                  399   100  300 300 900  900    900    900 300                                  430   100  100 300 300  300    300    300 300                                  773   100  100 300 300  300    300    300 300                                 __________________________________________________________________________      .sup.1,2 Top Dose = 300 mg/kg                                                  .sup.2 Low Dose = 11 mg/kg                                               

7.23.4. CONCLUSION

The toxicity profile of aprotinin analogs assessed by single-dose intravenous screening in Wistar rats was improved to a varying degree as compared to the toxicity profile of aprotinin. All aprotinin analogues had no-nephrotoxic-effect levels of 100 mg/kg or more as compared to 11 mg/kg for r-Aprotinin.

7.24. EXAMPLE 16: ELIMINATION AND DISTRIBUTION OF RECOMBINANT APROTININ AND APROTININ ANALOGS 7.24.1. MATERIALS

Recombinant authentic aprotinin and the analogues produced according to Examples 1-4 and 9-15 were dissolved in 0.9% NACl in order to obtain a dose volume of 1 μl/g rat. The concentrations of the injection solutions were controls analyzed by methods given in the method section.

7.24.2. METHODS

Female Wistar rats, weighing 200-230 g were used. Aprotinin and analogs were tested in two different models, 1) anaesthetized and 2) unanesthetized rats.

7.24.3. ANAESTHETIZED RATS

The rats were anaesthetized by intraperitoneal injection of pentobarbital sodium. The carotid artery and the jugular vein were exposed and cannulated with polyethylene catheters (PE-50, Intramedic). The carotid catheter was connected to a perfusor (B. Braun) for infusion of 3.8 ml 0.9% NaCl/h, and to a blood pressure transducer. Changes in blood pressure were recorded by using a chart recorder (Kipp & Zonen, BD 9). The analogues were administered as bolus injections over 15 seconds through the jugular catheter.

Blood samples were obtained from the carotid catheter at 3, 10, 20, 40, and 60 minutes after administration. The samples (0.45 ml) were collected in 3 ml test tubes containing 50 μl 10.13M sodium citrate and centrifuged. Plasma was stored at -20° C. until analysis. Sixty minutes after administration the rats were killed with an excessive dose of pentobarbital sodium and the kidneys and liver were removed, weighed, and stored at -80° C.

7.24.4. UNANESTHETIZED RATS

An oral dose of 2 ml distilled H₂ O was given prior to the administration of analogs. The analogs were given intravenously as bolus injections into a tail vein by using an intravenous catheter (Venflon 22 G, Viggo-Spectramed, Helsingborg, Sweden). After administration, the catheter was flushed by 0.5 ml 0.9% NACl and removed. A plaster was applied on the injection site in order to avoid bleeding from the tail. The rat was then placed in a metabolism cage in order to collect the urine produced.

After 3 hours the rat was killed by administration of CO₂ /O₂ (9/1) into the cage, and the kidneys and liver were removed and stored at -80° C. until analysis. During the CO₂ -administration, the rat emptied the urinary bladder and, after the rat was removed, the metabolism cage was rinsed with 0.9% NACl in order to obtain a total urine-NACl volume of 25 ml.

7.24.5. PREPARATION OF HOMOGENATES

One kidney (approx. 1 g) and approximately 2 g liver tissue were placed in separate 10 ml plastic test tubes and 2 ml 0.9% NACl were added. The tissues were homogenized 5 min by using a High Intensity Ultrasonic Processor (Model VC50, solics & Materials Inc. Danbury Conn., USA). The kidney and liver homogenates were then diluted with saline in order to obtain a total volume of 10-25 and 4 ml, respectively.

7.24.6. METHODS OF ANALYSIS

The levels of aprotinin and analogues in plasma, liver homogenates and injection solutions were measured photometrically on a Cobas Fara II (Roche). Briefly, plasma, homogenates or injection solutions were precipitated with acid in order to remove other kallikrein inhibitors than aprotinin. The kallikrein inhibitory activity in the sample were measured by using kallikrein from porcine pancreas (Sigma K 3627) and the chromogenic substrate S2266 (Kabi).

The levels in kidney homogenates and urine were measured by the same method, except for the precipitation step which was omitted, since the intrinsic kallikrein inhibitory activity in the diluted homogenates and urine was negligible.

Separate standard curves were employed for each analog in each medium.

7.24.7. STUDY DESIGN

14 groups of anaesthetized and 14 groups of unanesthetized rats were studied. A dose of 1.56 μmoles (approximately 10 mg) aprotinin or aprotinin analogue per kg body weight were administered to each rat. Basal data on the 28 groups are given in Table 8.

                  TABLE 8                                                          ______________________________________                                         Groups        n     BW         KW   LW                                         ______________________________________                                         rAprotinin A  5     251.8      0.98 9.9                                        KFN 1512 A    4     230.0      0.84 9.6                                        KFN 1514 A    4     220.5      0.85 8.6                                        KFN 1544 A    4     226.0      0.97 9.3                                        KFN 1545 A    4     224.8      0.92 9.2                                        KFN 1547 A    4     229.0      0.75 8.6                                        KFN 1660 A    4     220.5      0.93 9.7                                        KFN 1661 A    4     240.8      0.97 9.0                                        KFN 322 A     4     244.0      0.83 9.0                                        KFN 324 A     4     235.0      0.87 8.9                                        KFN 396 A     4     266.0      0.98 10.0                                       KFN 399 A     4     254.7      0.88 9.3                                        KFN 430 A     4     235.3      0.93 8.7                                        KFN 773 A     4     253.3      0.94 8.9                                        rAprotinin U  4     192.5      0.77 9.8                                        KFN 1512 U    5     191.0      0.65 7.2                                        KFN 1514 U    6     188.3      0.69 7.3                                        KFN 1544 U    5     190.0      0.64 6.5                                        KFN 1545 U    6     185.8      0.66 7.3                                        KFN 1547 U    5     188.0      0.67 7.3                                        KFN 1660 U    6     205.0      0.77 8.5                                        KFN 1661 U    6     204.2      0.80 8.1                                        KFN 322       6     193.3      0.71 8.7                                        KFN 324       4     198.8      0.76 9.5                                        KFN 396       5     194.0      0.77 10.7                                       KFN 399       5     183.0      0.74 9.5                                        KFN 430       4     195.0      0.77 8.9                                        KFN 773       5     193.0      0.75 9.8                                        ______________________________________                                          BW: Body weight (g). KW: Kidney weight (g) LW: Liver weight (g). A:            anaesthetized rat model U: Unanaesthetized rat model.                    

7.24.8. RESULTS 7.24.8.1.ANALOGS IN KIDNEYS AND URINE

The total content in kidneys (in percent of dosage) after 1 and 3 hours and in urine after 3 h is shown in FIGS. 12 and 13 and Table 9. It appears that great differences between the analogues were found.

As regards aprotinin, the content in kidneys 1 hour after administration was approximately 20% of the dose, whereas the content increased to more than 40% after 3 hours. The excretion of aprotinin in urine was negligible.

In order to evaluate whether the excretion in urine after 3 hours was associated with the net charge of the analogs, the degree of correlation between these figures was calculated. The content in urine was found to be strongly correlated with the net charges of the analogues (see Table 9).

                  TABLE 9                                                          ______________________________________                                                                Stab. index                                                                              Denat. tp.                                    Analogs    Acc. index  kidney    (°C.)                                  ______________________________________                                         rAprotinin A                                                                              2.10        0.90      100                                           KFN 1512 A 0.66        0.67      87                                            KFN 1514 A 1.32        0.87      88                                            KFN 1544 A 1.32        1.05      98                                            KFN 1545 A 1.99        0.71      93                                            KFN 1547 A 1.22        0.65      86                                            KFN 1660 A 0.50        0.55      77                                            KFN 1661 A 0.77        0.55      79                                            KFN 322 A  0.67        0.83      84                                            KFN 324 A  0.45        0.51      79                                            KFN 396 A  0.18        0.37      75                                            KFN 399 A  0.41        0.43      67                                            KFN 430 A  0.44        0.42      70                                            KFN 773 A  1.04        0.70      71                                            ______________________________________                                    

7.24.9. STABILITY OF ANALOGS

In order to study the stability of the analogs in kidney tissue, one kidney from 14 anaesthetized rats (one from each group) was divided into two pieces of identical weight. One piece was stored at 37° C. and the other piece at 4° C. After 4 hours, the tissues were homogenized and the content of analogues was measured. A stability index was defined as the content in the piece stored at 37° C. divided by the content in the piece stored at 4° C. The stability indices are given in Table 10 showing that rAprotinin, KFN 1514, KFN 1544 and KFN 322, seem to be the most stable compounds as compared to e.g. KFN 1660 and KFN 396 which appears to be more unstable.

The stability of analogues has also been studied by the determination of their denaturation temperature. The denaturation temperatures were found to be highly correlated with the content in kidney tissue 3 hours after administration (FIG. 14) and with the accumulation indices (FIG. 15), but not with urine excretion.

These data suggest that the net charge may be of importance for the excretion in urine, but of minor importance for the concentration and accumulation in kidneys. On the other hand, the renal accumulation seems to be related to the denaturation temperature and to the stability of analogues in kidney tissue.

It is, however, likely that the concentrations in kidney tissue, when measured 1 hour after administration, were changed due to degradation or redistribution. Thus, it is possible that the concentrations measured e.g. 10 min after administration would correlate with the net charge.

7.24.10. CONCLUSIONS

The following conclusions were made:

1) All analogs tested were taken up by the kidneys but to varying degrees. The accumulation in the kidneys seemed to be related to the thermostability and the stability in kidney tissue, but not to the net charge of the molecules.

2) The excretion in urine seemed to be associated with the net charge of the analogs, but not with the stability.

7.24.11. TOXICOLOGICAL SCREENING OF APROTININ ANALOGS BY SINGLE-DOSE INTRAVENOUS ADMINISTRATION TO WISTAR RATS 7.24.11.1. MATERIALS

The following aprotinin analogues with a reduced positive net charge and thermal stability compared to recombinant aprotinin (1-58) were selected for toxicological screening: KFN 322, KFN 324, KFN 396, KFN 399, KFN 430 and KFN 773. Their main characteristics as seen from a toxicological point of view, are shown in Table 6. Data for recombinant aprotinin are shown for comparison. The denaturation temperature is shown as an indication of biological stability.

7.24.11.2. DESIGN

On Day 1 of the screening of each analog, groups of 4 rats (3 for KFN 322) received 33, 300, or 900 mg analogue/kg body weight. Two control groups received physiological saline or physiological saline acidified with hydrochloric acid to an approximate pH 4.5. The latter solution served as vehicle. The dose volume was 10 ml/kg body weight in all cases. The rats were observed for 7 days and killed on Day 8. At autopsy the kidneys were weighed and prepared for histopathology. Response variables are shown in the heading of Table 10.

                  TABLE 10                                                         ______________________________________                                                           Macro-    Micro-                                                               scopic    scopic                                                     Mortality changes   changes                                            KFN-type                                                                               900 mg/kg 900 mg/kg 900 mg/kg                                                                               300 mg/kg                                 ______________________________________                                         322     3/3       PK 3/3    lethal   moderate                                  324     0/4       PK 3/4    NF       moderate                                  396     0/4       normal    weak     normal                                    399     0/4       normal    normal   normal                                    430     4/4       PK 2/2    lethal   normal                                    773     4/4       normal    lethal   normal                                    ______________________________________                                    

7.24.12. RESULTS

Results from individual screenings are summarized in Table 10. By way of comparison, rAprotinin (1-58) has a mortality dose of 300 mg/kg, macroscopic kidney changes at 33 mg/kg and microscopic kidney changes at 11 mg/kg.

No histopathological kidney change was seen after administration (300 mg/kg body weight) of the analogues except KFN 322 and KFN 324. Furthermore, no histopathological kidney change was seen after administration of 900 mg/kg body weight of KFN 399.

7.24.13. CONCLUSION

The toxicity profile of aprotinin analogues assessed by single-dose intravenous screening in Wistar rats was improved to a varying degree as compared to the toxicity profile of aprotinin.

The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the are from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

    __________________________________________________________________________     SEQUENCE LISTING                                                               (1) GENERAL INFORMATION:                                                       (iii) NUMBER OF SEQUENCES: 83                                                  (2) INFORMATION FOR SEQ ID NO:1:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Bovine                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                        ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                        XaaAspPheCysLeuGluProProTyrThrXaaXaaXaaXaaXaaXaa                               151015                                                                         XaaXaaArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPhe                               202530                                                                         ValTyrGlyGlyXaaArgAlaXaaXaaAsnAsnPheLysSerAlaGlu                               354045                                                                         AspCysMetArgThrCysGlyGlyAla                                                    5055                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                        XaaAspPheCysLeuGluProProTyrThrGlyProCysXaaXaaXaa                               151015                                                                         XaaXaaArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPhe                               202530                                                                         ValTyrGlyGlyCysArgAlaXaaXaaAsnAsnPheLysSerAlaGlu                               354045                                                                         AspCysMetArgThrCysGlyGlyAla                                                    5055                                                                           (2) INFORMATION FOR SEQ ID NO:4:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                        XaaAspPheCysLeuGluProProTyrThrGlyProCysLysXaaXaa                               151015                                                                         XaaXaaArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPhe                               202530                                                                         ValTyrGlyGlyCysArgAlaXaaXaaAsnAsnPheLysSerAlaGlu                               354045                                                                         AspCysMetArgThrCysGlyGlyAla                                                    5055                                                                           (2) INFORMATION FOR SEQ ID NO:5:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                        AspPheCysLeuGluProProTyrThrGlyProCysLysAlaAlaIle                               151015                                                                         IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal                               202530                                                                         TyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAlaGluAsp                               354045                                                                         CysMetArgThrCysGlyGlyAla                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                        AspPheCysLeuGluProProTyrThrGlyProCysLysAlaAlaIle                               151015                                                                         GluArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal                               202530                                                                         TyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAlaGluAsp                               354045                                                                         CysMetArgThrCysGlyGlyAla                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:7:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                        AspPheCysLeuGluProProTyrThrGlyProCysArgAlaAlaIle                               151015                                                                         IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal                               202530                                                                         TyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAlaGluAsp                               354045                                                                         CysMetArgThrCysGlyGlyAla                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                        AspPheCysLeuGluProProTyrThrGlyProCysLysAlaAlaIle                               151015                                                                         IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal                               202530                                                                         TyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAlaGluAsp                               354045                                                                         CysMetArgThrCysGlyGlyAla                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:9:                                               (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                        AspPheCysLeuGluProProTyrThrGlyProCysLysAlaAlaIle                               151015                                                                         GluArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal                               202530                                                                         TyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAlaGluAsp                               354045                                                                         CysMetArgThrCysGlyGlyAla                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:10:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                       AspPheCysLeuGluProProTyrThrGlyProCysArgAlaAlaIle                               151015                                                                         IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal                               202530                                                                         TyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAlaGluAsp                               354045                                                                         CysMetArgThrCysGlyGlyAla                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:11:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                       ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         AlaIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                       ArgProAspPheCysLeuGluProProTyrThrGlyProCysArgAla                               151015                                                                         AlaIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                       ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         AlaIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                       ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         AlaIleGluArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:15:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                       ArgProAspPheCysLeuGluProProTyrThrGlyProCysArgAla                               151015                                                                         AlaIleGluArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:16:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                       XaaAspPheCysLeuGluProProXaaThrGlyProCysLysAlaArg                               151015                                                                         IleIleXaaTyrPheTyrXaaAlaXaaAlaGlyLeuCysXaaThrPhe                               202530                                                                         ValTyrGlyGlyCysArgXaaXaaXaaAsnXaaPheXaaSerAlaGlu                               354045                                                                         AspCysMetXaaThrCysGlyGlyAla                                                    5055                                                                           (2) INFORMATION FOR SEQ ID NO:17:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 57 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                       XaaAspPheCysLeuGluProProXaaThrGlyProCysXaaXaaXaa                               151015                                                                         XaaXaaXaaTyrPheTyrXaaAlaXaaAlaGlyLeuCysXaaThrPhe                               202530                                                                         XaaTyrXaaGlyCysXaaXaaXaaXaaAsnXaaPheXaaSerAlaGlu                               354045                                                                         AspCysMetXaaThrCysGlyGlyAla                                                    5055                                                                           (2) INFORMATION FOR SEQ ID NO:18:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 37 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                       AAAGAGATTTCTGTTTGGAACCTCCATACACTGGTCC37                                        (2) INFORMATION FOR SEQ ID NO:19:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 38 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                       TTACATGGACCAGTGTATGGAGGTTCCAAACAGAAACT38                                       (2) INFORMATION FOR SEQ ID NO:20:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 35 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                       ATGTAAAGCTAGAATCATCAGATACTTCTACAACG35                                          (2) INFORMATION FOR SEQ ID NO:21:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 34 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                       TTCGGCGTTGTAGAAGTATCTGATGATTCTAGCT34                                           (2) INFORMATION FOR SEQ ID NO:22:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                       CCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCT39                                      (2) INFORMATION FOR SEQ ID NO:23:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 40 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                       CTCTGCAGCCACCGTAAACGAAAGTTTGACACAAACCAGC40                                     (2) INFORMATION FOR SEQ ID NO:24:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                       GCAGAGCTAAGTCCAACAACTTCAAGT27                                                  (2) INFORMATION FOR SEQ ID NO:25:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                       AGCAGACTTGAAGTTGTTGGACTTAG26                                                   (2) INFORMATION FOR SEQ ID NO:26:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                       CTGCTGAAGACTGCATGAGAACTTGTGGTGGTGCCTAAT39                                      (2) INFORMATION FOR SEQ ID NO:27:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 38 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                       CTAGATTAGGCACCACCACAAGTTCTCATGCAGTCTTC38                                       (2) INFORMATION FOR SEQ ID NO:28:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 177 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 6..173                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                       AAAGAGATTTCTGTTTGGAACCTCCATACACTGGTCCATGTAAAGCT47                              AspPheCysLeuGluProProTyrThrGlyProCysLysAla                                     1510                                                                           AGAATCATCAGATACTTCTACAACGCCAAGGCTGGTTTGTGTCAAACT95                             ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               15202530                                                                       TTCGTTTACGGTGGCTGCAGAGCTAAGAGAAACAACTTCAAGTCTGCT143                            PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GAAGACTGCATGAGAACTTGTGGTGGTGCCTAAT177                                          GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:29:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                       AspPheCysLeuGluProProTyrThrGlyProCysLysAlaArgIle                               151015                                                                         IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal                               202530                                                                         TyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAlaGluAsp                               354045                                                                         CysMetArgThrCysGlyGlyAla                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:30:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 18 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                       AspPheCysLeuGluProProTyrThrGlyProCysLysAlaArgIle                               151015                                                                         IleArg                                                                         (2) INFORMATION FOR SEQ ID NO:31:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                       GCAGAGCTAAGTCCAACAACTTCAAGT27                                                  (2) INFORMATION FOR SEQ ID NO:32:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                       AGCAGACTTGAAGTTGTTGGACTTAG26                                                   (2) INFORMATION FOR SEQ ID NO:33:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 177 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 6..173                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                       AAAGAGATTTCTGTTTGGAACCTCCATACACTGGTCCATGTAAAGCT47                              AspPheCysLeuGluProProTyrThrGlyProCysLysAla                                     1510                                                                           AGAATCATCAGATACTTCTACAACGCCAAGGCTGGTTTGTGTCAAACT95                             ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               15202530                                                                       TTCGTTTACGGTGGCTGCAGAGCTAAGTCCAACAACTTCAAGTCTGCT143                            PheValTyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAla                               354045                                                                         GAAGACTGCATGAGAACTTGTGGTGGTGCCTAAT177                                          GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:34:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 56 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                       AspPheCysLeuGluProProTyrThrGlyProCysLysAlaArgIle                               151015                                                                         IleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThrPheVal                               202530                                                                         TyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAlaGluAsp                               354045                                                                         CysMetArgThrCysGlyGlyAla                                                       5055                                                                           (2) INFORMATION FOR SEQ ID NO:35:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 20 amino acids                                                     (B) TYPE: amino acid                                                           (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                       AspPheCysLeuGluProProTyrThrGlyProCysLysAlaArgIle                               151015                                                                         IleArgTyrPhe                                                                   20                                                                             (2) INFORMATION FOR SEQ ID NO:36:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 43 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                       CTGGTCCATGTAAAGCTGCTATCATCAGATACTTCTACAACGC43                                  (2) INFORMATION FOR SEQ ID NO:37:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 50 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                       CTTGGCGTTGTAGAAGTATCTGATGATAGCAGCTTTACATGGACCAGTGT50                           (2) INFORMATION FOR SEQ ID NO:38:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 178 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..174                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                       AGGCCTGATTTCTGTTTGGAACCTCCATACACTGGTCCATGTAAAGCT48                             ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         AGAATCATCAGATACTTCTACAACGCCAAGGCTGGTTTGTGTCAAACT96                             ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         TTCGTTTACGGTGGCTGCAGAGCTAAGAGAAACAACTTCAAGTCTGCT144                            PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GAAGACTGCATGAGAACTTGTGGTGGTGCCTAAT178                                          GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:39:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                       ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysArgAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:40:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 178 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 1..174                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                       AGGCCTGATTTCTGTTTGGAACCTCCATACACTGGTCCATGTAAAGCT48                             ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         AGAATCATCAGATACTTCTACAACGCCAAGGCTGGTTTGTGTCAAACT96                             ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         TTCGTTTACGGTGGCTGCAGAGCTAAGTCCAACAACTTCAAGTCTGCT144                            PheValTyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAla                               354045                                                                         GAAGACTGCATGAGAACTTGTGGTGGTGCCTAAT178                                          GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:41:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 58 amino acids                                                     (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                       ArgProAspPheCysLeuGluProProTyrThrGlyProCysLysAla                               151015                                                                         ArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeuCysGlnThr                               202530                                                                         PheValTyrGlyGlyCysArgAlaLysSerAsnAsnPheLysSerAla                               354045                                                                         GluAspCysMetArgThrCysGlyGlyAla                                                 5055                                                                           (2) INFORMATION FOR SEQ ID NO:42:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 43 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                       CTGGTCCATGTAAAGCTGCTATCGAAAGATACTTCTACAACGC43                                  (2) INFORMATION FOR SEQ ID NO:43:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 50 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                       CTTGGCGTTGTAGAAGTATCTTTCGATAGCAGCTTTACATGGACCAGTGT50                           (2) INFORMATION FOR SEQ ID NO:44:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 43 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                       CTGGTCCATGTAGAGCTGCTATCATCAGATACTTCTACAACGC43                                  (2) INFORMATION FOR SEQ ID NO:45:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 50 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                       CTTGGCGTTGTAGAAGTATCTGATGATAGCAGCTCTACATGGACCAGTGT50                           (2) INFORMATION FOR SEQ ID NO:46:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 63 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                       CATGGCTGAGAGATTGGAGAAGAGAGAGCCTGATTTCTGTTTGGAACCTCCATACACTGG60                 TCC63                                                                          (2) INFORMATION FOR SEQ ID NO:47:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 65 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                       TTACATGGACCAGTGTATGGAGGTTCCAAACAGAAATCAGGCTCTCTCTTCTCCAATCTC60                 TCAGC65                                                                        (2) INFORMATION FOR SEQ ID NO:48:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 39 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                       CCGAAGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCT39                                      (2) INFORMATION FOR SEQ ID NO:49:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 40 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                       CTCTGCAGCCACCGTAAACGAAAGTTTGACACAAACCAGC40                                     (2) INFORMATION FOR SEQ ID NO:50:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 27 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                       GCAGAGCTGAAAGAAACAACTTCGAAT27                                                  (2) INFORMATION FOR SEQ ID NO:51:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 26 base pairs                                                      (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                       AGCAGATTCGAAGTTGTTTCTTTCAG26                                                   (2) INFORMATION FOR SEQ ID NO:52:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 418 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..235                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 236..409                                                         (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..409                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGACCAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATC109                           MetLysAlaValPheLeuValLeuSerLeuIle                                              53-50-45                                                                       GGATTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAG157                            GlyPheCysTrpAlaGlnProValThrGlyAspGluSerSerValGlu                               40-35-30                                                                       ATTCCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAAC205                            IleProGluGluSerLeuIleIleAlaGluAsnThrThrLeuAlaAsn                               25-20-15                                                                       GTCGCCATGGCTGAGAGATTGGAGAAGAGAGAGCCTGATTTCTGTTTG253                            ValAlaMetAlaGluArgLeuGluLysArgGluProAspPheCysLeu                               10-515                                                                         GAACCTCCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTC301                            GluProProTyrThrGlyProCysLysAlaArgIleIleArgTyrPhe                               101520                                                                         TACAACGCCGAAGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGC349                            TyrAsnAlaGluAlaGlyLeuCysGlnThrPheValTyrGlyGlyCys                               253035                                                                         AGAGCTGAAAGAAACAACTTCGAATCTGCTGAAGACTGCATGAGAACT397                            ArgAlaGluArgAsnAsnPheGluSerAlaGluAspCysMetArgThr                               404550                                                                         TGTGGTGGTGCCTAATCTAGA418                                                       CysGlyGlyAla                                                                   55                                                                             (2) INFORMATION FOR SEQ ID NO:53:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 111 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                       MetLysAlaValPheLeuValLeuSerLeuIleGlyPheCysTrpAla                               53-50-45- 40                                                                   GlnProValThrGlyAspGluSerSerValGluIleProGluGluSer                               35-30-25                                                                       LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaGlu                               20-15-10                                                                       ArgLeuGluLysArgGluProAspPheCysLeuGluProProTyrThr                               51510                                                                          GlyProCysLysAlaArgIleIleArgTyrPheTyrAsnAlaGluAla                               152025                                                                         GlyLeuCysGlnThrPheValTyrGlyGlyCysArgAlaGluArgAsn                               303540                                                                         AsnPheGluSerAlaGluAspCysMetArgThrCysGlyGlyAla                                  455055                                                                         (2) INFORMATION FOR SEQ ID NO:54:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 418 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..409                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..235                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 236..409                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGACCAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATC109                           MetLysAlaValPheLeuValLeuSerLeuIle                                              53-50-45                                                                       GGATTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAG157                            GlyPheCysTrpAlaGlnProValThrGlyAspGluSerSerValGlu                               40-35-30                                                                       ATTCCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAAC205                            IleProGluGluSerLeuIleIleAlaGluAsnThrThrLeuAlaAsn                               25-20-15                                                                       GTCGCCATGGCTGAGAGATTGGAGAAGAGAGAGCCTGATTTCTGTTTG253                            ValAlaMetAlaGluArgLeuGluLysArgGluProAspPheCysLeu                               10-515                                                                         GAACCTCCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTC301                            GluProProTyrThrGlyProCysLysAlaArgIleIleArgTyrPhe                               101520                                                                         TACAACGCCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGC349                            TyrAsnAlaLysAlaGlyLeuCysGlnThrPheValTyrGlyGlyCys                               253035                                                                         AGAGCTAAGGAAAACAACTTCGAATCTGCTGAAGACTGCATGAGAACT397                            ArgAlaLysGluAsnAsnPheGluSerAlaGluAspCysMetArgThr                               404550                                                                         TGTGGTGGTGCCTAATCTAGA418                                                       CysGlyGlyAla                                                                   55                                                                             (2) INFORMATION FOR SEQ ID NO:55:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 111 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                       MetLysAlaValPheLeuValLeuSerLeuIleGlyPheCysTrpAla                               53-50-45- 40                                                                   GlnProValThrGlyAspGluSerSerValGluIleProGluGluSer                               35-30-25                                                                       LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaGlu                               20-15-10                                                                       ArgLeuGluLysArgGluProAspPheCysLeuGluProProTyrThr                               51510                                                                          GlyProCysLysAlaArgIleIleArgTyrPheTyrAsnAlaLysAla                               152025                                                                         GlyLeuCysGlnThrPheValTyrGlyGlyCysArgAlaLysGluAsn                               303540                                                                         AsnPheGluSerAlaGluAspCysMetArgThrCysGlyGlyAla                                  455055                                                                         (2) INFORMATION FOR SEQ ID NO:56:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 418 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..409                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..235                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 236..409                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGACCAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATC109                           MetLysAlaValPheLeuValLeuSerLeuIle                                              53-50-45                                                                       GGATTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAG157                            GlyPheCysTrpAlaGlnProValThrGlyAspGluSerSerValGlu                               40-35-30                                                                       ATTCCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAAC205                            IleProGluGluSerLeuIleIleAlaGluAsnThrThrLeuAlaAsn                               25-20-15                                                                       GTCGCCATGGCTGAGAGATTGGAGAAGAGAAGGCCTGATTTCTGTTTG253                            ValAlaMetAlaGluArgLeuGluLysArgArgProAspPheCysLeu                               10-515                                                                         GAACCTCCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTC301                            GluProProTyrThrGlyProCysLysAlaArgIleIleArgTyrPhe                               101520                                                                         TACAACGCCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGC349                            TyrAsnAlaLysAlaGlyLeuCysGlnThrPheValTyrGlyGlyCys                               253035                                                                         AGAGCTAAGGAAAACAACTTCGAATCTGCTGAAGACTGCATGAGAACT397                            ArgAlaLysGluAsnAsnPheGluSerAlaGluAspCysMetArgThr                               404550                                                                         TGTGGTGGTGCCTAATCTAGA418                                                       CysGlyGlyAla                                                                   55                                                                             (2) INFORMATION FOR SEQ ID NO:57:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 111 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                       MetLysAlaValPheLeuValLeuSerLeuIleGlyPheCysTrpAla                               53-50-45- 40                                                                   GlnProValThrGlyAspGluSerSerValGluIleProGluGluSer                               35-30-25                                                                       LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaGlu                               20-15-10                                                                       ArgLeuGluLysArgArgProAspPheCysLeuGluProProTyrThr                               51510                                                                          GlyProCysLysAlaArgIleIleArgTyrPheTyrAsnAlaLysAla                               152025                                                                         GlyLeuCysGlnThrPheValTyrGlyGlyCysArgAlaLysGluAsn                               303540                                                                         AsnPheGluSerAlaGluAspCysMetArgThrCysGlyGlyAla                                  455055                                                                         (2) INFORMATION FOR SEQ ID NO:58:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 418 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..409                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..235                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 236..409                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGACCAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATC109                           MetLysAlaValPheLeuValLeuSerLeuIle                                              1510                                                                           GGATTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAG157                            GlyPheCysTrpAlaGlnProValThrGlyAspGluSerSerValGlu                               152025                                                                         ATTCCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAAC205                            IleProGluGluSerLeuIleIleAlaGluAsnThrThrLeuAlaAsn                               303540                                                                         GTCGCCATGGCTGAGAGATTGGAGAAGAGAAGGCCTGATTTCTGTTTG253                            ValAlaMetAlaGluArgLeuGluLysArgArgProAspPheCysLeu                               455055                                                                         GAACCTCCATCTACTGGTCCATGTAAAGCTAGAATCATCAGATACTTC301                            GluProProSerThrGlyProCysLysAlaArgIleIleArgTyrPhe                               60657075                                                                       TACGACGCCACTGCTGGTTTGTGTGAAACTTTCGTTTACGGTGGCTGC349                            TyrAspAlaThrAlaGlyLeuCysGluThrPheValTyrGlyGlyCys                               808590                                                                         AGAGCTAACAGAAACAACTTCAAGTCTGCTGAAGACTGCATGGAAACT397                            ArgAlaAsnArgAsnAsnPheLysSerAlaGluAspCysMetGluThr                               95100105                                                                       TGTGGTGGTGCCTAATCTAGA418                                                       CysGlyGlyAla                                                                   110                                                                            (2) INFORMATION FOR SEQ ID NO:59:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 111 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                       MetLysAlaValPheLeuValLeuSerLeuIleGlyPheCysTrpAla                               151015                                                                         GlnProValThrGlyAspGluSerSerValGluIleProGluGluSer                               202530                                                                         LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaGlu                               354045                                                                         ArgLeuGluLysArgArgProAspPheCysLeuGluProProSerThr                               505560                                                                         GlyProCysLysAlaArgIleIleArgTyrPheTyrAspAlaThrAla                               65707580                                                                       GlyLeuCysGluThrPheValTyrGlyGlyCysArgAlaAsnArgAsn                               859095                                                                         AsnPheLysSerAlaGluAspCysMetGluThrCysGlyGlyAla                                  100105110                                                                      (2) INFORMATION FOR SEQ ID NO:60:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 418 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..409                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..235                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 236..409                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGACCAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATC109                           MetLysAlaValPheLeuValLeuSerLeuIle                                              53-50-45                                                                       GGATTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAG157                            GlyPheCysTrpAlaGlnProValThrGlyAspGluSerSerValGlu                               40-35-30                                                                       ATTCCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAAC205                            IleProGluGluSerLeuIleIleAlaGluAsnThrThrLeuAlaAsn                               25-20-15                                                                       GTCGCCATGGCTGAGAGATTGGAGAAGAGAAGGCCTGATTTCTGTTTG253                            ValAlaMetAlaGluArgLeuGluLysArgArgProAspPheCysLeu                               10-515                                                                         GAACCTCCATCTACTGGTCCATGTAAAGCTAGAATCATCTTGTACTTC301                            GluProProSerThrGlyProCysLysAlaArgIleIleLeuTyrPhe                               101520                                                                         TACAACGCCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGC349                            TyrAsnAlaLysAlaGlyLeuCysGlnThrPheValTyrGlyGlyCys                               253035                                                                         AGAGGTAACGGTAACCAATTCTACTCTGCTGAAGACTGCATGAGAACT397                            ArgGlyAsnGlyAsnGlnPheTyrSerAlaGluAspCysMetArgThr                               404550                                                                         TGTGGTGGTGCCTAATCTAGA418                                                       CysGlyGlyAla                                                                   55                                                                             (2) INFORMATION FOR SEQ ID NO:61:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 111 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                       MetLysAlaValPheLeuValLeuSerLeuIleGlyPheCysTrpAla                               53-50-45- 40                                                                   GlnProValThrGlyAspGluSerSerValGluIleProGluGluSer                               35-30-25                                                                       LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaGlu                               20-15-10                                                                       ArgLeuGluLysArgArgProAspPheCysLeuGluProProSerThr                               51510                                                                          GlyProCysLysAlaArgIleIleLeuTyrPheTyrAsnAlaLysAla                               152025                                                                         GlyLeuCysGlnThrPheValTyrGlyGlyCysArgGlyAsnGlyAsn                               303540                                                                         GlnPheTyrSerAlaGluAspCysMetArgThrCysGlyGlyAla                                  455055                                                                         (2) INFORMATION FOR SEQ ID NO:62:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 110 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                       CAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCTAAGTCCAACAACTT60                 CGAATCTGCTGAAGACTGCATGAGAACTTGTGGTGGTGCCTAATCTAGAG110                          (2) INFORMATION FOR SEQ ID NO:63:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 110 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                       TCGACTCTAGATTAGGCACCACCACAAGTTCTCATGCAGTCTTCAGCAGATTCGAAGTTG60                 TTGGACTTAGCTCTGCAGCCACCGTAAACGAAAGTTTGACACAAACCAGC110                          (2) INFORMATION FOR SEQ ID NO:64:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 508 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..499                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..331                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 332..499                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGATTAAAAGAATGAGATTTCCTTCAATTTTTACTGCAGTTTTA109                           MetArgPheProSerIlePheThrAlaValLeu                                              85-80-75                                                                       TTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAA157                            PheAlaAlaSerSerAlaLeuAlaAlaProValAsnThrThrThrGlu                               70-65-60                                                                       GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGAT205                            AspGluThrAlaGlnIleProAlaGluAlaValIleGlyTyrSerAsp                               55-50- 45                                                                      TTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACA253                            LeuGluGlyAspPheAspValAlaValLeuProPheSerAsnSerThr                               40-35-30                                                                       AATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCT301                            AsnAsnGlyLeuLeuPheIleAsnThrThrIleAlaSerIleAlaAla                               25-20-15                                                                       AAAGAAGAAGGGGTATCTTTGGATAAAAGAGATTTCTGTTTGGAACCT349                            LysGluGluGlyValSerLeuAspLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACAAC397                            ProTyrThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsn                               101520                                                                         GCCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCT445                            AlaLysAlaGlyLeuCysGlnThrPheValTyrGlyGlyCysArgAla                               253035                                                                         AAGTCCAACAACTTCGAATCTGCTGAAGACTGCATGAGAACTTGTGGT493                            LysSerAsnAsnPheGluSerAlaGluAspCysMetArgThrCysGly                               404550                                                                         GGTGCCTAATCTAGA508                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:65:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 141 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                       MetArgPheProSerIlePheThrAlaValLeuPheAlaAlaSerSer                               85-80-75-70                                                                    AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                               65-60-55                                                                       IleProAlaGluAlaValIleGlyTyrSerAspLeuGluGlyAspPhe                               50-45- 40                                                                      AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                               35-30-25                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                               20-15-10                                                                       SerLeuAspLysArgAspPheCysLeuGluProProTyrThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeu                               152025                                                                         CysGlnThrPheValTyrGlyGlyCysArgAlaLysSerAsnAsnPhe                               303540                                                                         GluSerAlaGluAspCysMetArgThrCysGlyGlyAla                                        455055                                                                         (2) INFORMATION FOR SEQ ID NO:66:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 110 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                       CAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCTAAGTCCAACAACTT60                 CGCTTCTGCTGAAGACTGCATGAGAACTTGTGGTGGTGCCTAATCTAGAG110                          (2) INFORMATION FOR SEQ ID NO:67:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 110 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                       TCGACTCTAGATTAGGCACCACCACAAGTTCTCATGCAGTCTTCAGCAGAAGCGAAGTTG60                 TTGGACTTAGCTCTGCAGCCACCGTAAACGAAAGTTTGACACAAACCAGC110                          (2) INFORMATION FOR SEQ ID NO:68:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 508 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..499                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..331                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 332..499                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGATTAAAAGAATGAGATTTCCTTCAATTTTTACTGCAGTTTTA109                           MetArgPheProSerIlePheThrAlaValLeu                                              85-80-75                                                                       TTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAA157                            PheAlaAlaSerSerAlaLeuAlaAlaProValAsnThrThrThrGlu                               70-65-60                                                                       GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGAT205                            AspGluThrAlaGlnIleProAlaGluAlaValIleGlyTyrSerAsp                               55-50- 45                                                                      TTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACA253                            LeuGluGlyAspPheAspValAlaValLeuProPheSerAsnSerThr                               40-35-30                                                                       AATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCT301                            AsnAsnGlyLeuLeuPheIleAsnThrThrIleAlaSerIleAlaAla                               25-20-15                                                                       AAAGAAGAAGGGGTATCTTTGGATAAAAGAGATTTCTGTTTGGAACCT349                            LysGluGluGlyValSerLeuAspLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACAAC397                            ProTyrThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsn                               101520                                                                         GCCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCT445                            AlaLysAlaGlyLeuCysGlnThrPheValTyrGlyGlyCysArgAla                               253035                                                                         AAGTCCAACAACTTCGCTTCTGCTGAAGACTGCATGAGAACTTGTGGT493                            LysSerAsnAsnPheAlaSerAlaGluAspCysMetArgThrCysGly                               404550                                                                         GGTGCCTAATCTAGA508                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:69:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 141 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                       MetArgPheProSerIlePheThrAlaValLeuPheAlaAlaSerSer                               85-80-75-70                                                                    AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                               65-60-55                                                                       IleProAlaGluAlaValIleGlyTyrSerAspLeuGluGlyAspPhe                               50-45- 40                                                                      AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                               35-30-25                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                               20-15-10                                                                       SerLeuAspLysArgAspPheCysLeuGluProProTyrThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeu                               152025                                                                         CysGlnThrPheValTyrGlyGlyCysArgAlaLysSerAsnAsnPhe                               303540                                                                         AlaSerAlaGluAspCysMetArgThrCysGlyGlyAla                                        455055                                                                         (2) INFORMATION FOR SEQ ID NO:70:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 412 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..403                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..235                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 236..403                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGACCAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATC109                           MetLysAlaValPheLeuValLeuSerLeuIle                                              53-50-45                                                                       GGATTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAG157                            GlyPheCysTrpAlaGlnProValThrGlyAspGluSerSerValGlu                               40-35-30                                                                       ATTCCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAAC205                            IleProGluGluSerLeuIleIleAlaGluAsnThrThrLeuAlaAsn                               25-20-15                                                                       GTCGCCATGGCTGAGAGATTGGAGAAGAGGGATTTCTGTTTGGAACCT253                            ValAlaMetAlaGluArgLeuGluLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATCTACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACGAC301                            ProSerThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsp                               101520                                                                         GCCACTGCTGGTTTGTGTGAAACTTTCGTTTACGGTGGCTGCAGAGCT349                            AlaThrAlaGlyLeuCysGluThrPheValTyrGlyGlyCysArgAla                               253035                                                                         AACAGAAACAACTTCAAGTCTGCTGAAGACTGCATGGAAACTTGTGGT397                            AsnArgAsnAsnPheLysSerAlaGluAspCysMetGluThrCysGly                               404550                                                                         GGTGCCTAATCTAGA412                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:71:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 109 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                       MetLysAlaValPheLeuValLeuSerLeuIleGlyPheCysTrpAla                               53-50-45- 40                                                                   GlnProValThrGlyAspGluSerSerValGluIleProGluGluSer                               35-30-25                                                                       LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaGlu                               20-15-10                                                                       ArgLeuGluLysArgAspPheCysLeuGluProProSerThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAspAlaThrAlaGlyLeu                               152025                                                                         CysGluThrPheValTyrGlyGlyCysArgAlaAsnArgAsnAsnPhe                               303540                                                                         LysSerAlaGluAspCysMetGluThrCysGlyGlyAla                                        455055                                                                         (2) INFORMATION FOR SEQ ID NO:72:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 508 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..499                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..331                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 332..499                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGATTAAAAGAATGAGATTTCCTTCAATTTTTACTGCAGTTTTA109                           MetArgPheProSerIlePheThrAlaValLeu                                              85-80-75                                                                       TTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAA157                            PheAlaAlaSerSerAlaLeuAlaAlaProValAsnThrThrThrGlu                               70-65-60                                                                       GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGAT205                            AspGluThrAlaGlnIleProAlaGluAlaValIleGlyTyrSerAsp                               55-50- 45                                                                      TTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACA253                            LeuGluGlyAspPheAspValAlaValLeuProPheSerAsnSerThr                               40-35-30                                                                       AATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCT301                            AsnAsnGlyLeuLeuPheIleAsnThrThrIleAlaSerIleAlaAla                               25-20-15                                                                       AAAGAAGAAGGGGTATCTTTGGATAAAAGAGATTTCTGTTTGGAACCT349                            LysGluGluGlyValSerLeuAspLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACGAC397                            ProTyrThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsp                               101520                                                                         GCCACTGCTGGTTTGTGTGAAACTTTCGTTTACGGTGGCTGCAGAGCT445                            AlaThrAlaGlyLeuCysGluThrPheValTyrGlyGlyCysArgAla                               253035                                                                         AAGAGAAACAACTTCAAGTCTGCTGAAGACTGCATGGAAACTTGTGGT493                            LysArgAsnAsnPheLysSerAlaGluAspCysMetGluThrCysGly                               404550                                                                         GGTGCCTAATCTAGA508                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:73:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 141 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                       MetArgPheProSerIlePheThrAlaValLeuPheAlaAlaSerSer                               85-80-75-70                                                                    AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                               65-60-55                                                                       IleProAlaGluAlaValIleGlyTyrSerAspLeuGluGlyAspPhe                               50-45- 40                                                                      AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                               35-30-25                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                               20-15-10                                                                       SerLeuAspLysArgAspPheCysLeuGluProProTyrThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAspAlaThrAlaGlyLeu                               152025                                                                         CysGluThrPheValTyrGlyGlyCysArgAlaLysArgAsnAsnPhe                               303540                                                                         LysSerAlaGluAspCysMetGluThrCysGlyGlyAla                                        455055                                                                         (2) INFORMATION FOR SEQ ID NO:74:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 412 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..403                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..235                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 236..403                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGACCAAAAGAATGAAGGCTGTTTTCTTGGTTTTGTCCTTGATC109                           MetLysAlaValPheLeuValLeuSerLeuIle                                              53-50-45                                                                       GGATTCTGCTGGGCCCAACCAGTCACTGGCGATGAATCATCTGTTGAG157                            GlyPheCysTrpAlaGlnProValThrGlyAspGluSerSerValGlu                               40-35-30                                                                       ATTCCGGAAGAGTCTCTGATCATCGCTGAAAACACCACTTTGGCTAAC205                            IleProGluGluSerLeuIleIleAlaGluAsnThrThrLeuAlaAsn                               25-20-15                                                                       GTCGCCATGGCTGAGAGATTGGAGAAGAGGGATTTCTGTTTGGAACCT253                            ValAlaMetAlaGluArgLeuGluLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATCTACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACAAC301                            ProSerThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsn                               101520                                                                         GCCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGGT349                            AlaLysAlaGlyLeuCysGlnThrPheValTyrGlyGlyCysArgGly                               253035                                                                         AACGGCAACAACTTCAAGTCTGCTGAAGACTGCATGGAAACTTGTGGT397                            AsnGlyAsnAsnPheLysSerAlaGluAspCysMetGluThrCysGly                               404550                                                                         GGTGCCTAATCTAGA412                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:75:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 109 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                       MetLysAlaValPheLeuValLeuSerLeuIleGlyPheCysTrpAla                               53-50-45- 40                                                                   GlnProValThrGlyAspGluSerSerValGluIleProGluGluSer                               35-30-25                                                                       LeuIleIleAlaGluAsnThrThrLeuAlaAsnValAlaMetAlaGlu                               20-15-10                                                                       ArgLeuGluLysArgAspPheCysLeuGluProProSerThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeu                               152025                                                                         CysGlnThrPheValTyrGlyGlyCysArgGlyAsnGlyAsnAsnPhe                               303540                                                                         LysSerAlaGluAspCysMetGluThrCysGlyGlyAla                                        455055                                                                         (2) INFORMATION FOR SEQ ID NO:76:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 508 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..499                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..331                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 332..499                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGATTAAAAGAATGAGATTTCCTTCAATTTTTACTGCAGTTTTA109                           MetArgPheProSerIlePheThrAlaValLeu                                              85-80-75                                                                       TTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAA157                            PheAlaAlaSerSerAlaLeuAlaAlaProValAsnThrThrThrGlu                               70-65-60                                                                       GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGAT205                            AspGluThrAlaGlnIleProAlaGluAlaValIleGlyTyrSerAsp                               55-50- 45                                                                      TTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACA253                            LeuGluGlyAspPheAspValAlaValLeuProPheSerAsnSerThr                               40-35-30                                                                       AATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCT301                            AsnAsnGlyLeuLeuPheIleAsnThrThrIleAlaSerIleAlaAla                               25-20-15                                                                       AAAGAAGAAGGGGTATCTTTGGATAAAAGAGATTTCTGTTTGGAACCT349                            LysGluGluGlyValSerLeuAspLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACAAC397                            ProTyrThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsn                               101520                                                                         GCCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCT445                            AlaLysAlaGlyLeuCysGlnThrPheValTyrGlyGlyCysArgAla                               253035                                                                         AAGTCCAACAACTTCAAGTCTGCTGAAGACTGCATGGAAACTTGTGGT493                            LysSerAsnAsnPheLysSerAlaGluAspCysMetGluThrCysGly                               404550                                                                         GGTGCCTAATCTAGA508                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:77:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 141 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                       MetArgPheProSerIlePheThrAlaValLeuPheAlaAlaSerSer                               85-80-75-70                                                                    AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                               65-60-55                                                                       IleProAlaGluAlaValIleGlyTyrSerAspLeuGluGlyAspPhe                               50-45- 40                                                                      AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                               35-30-25                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                               20-15-10                                                                       SerLeuAspLysArgAspPheCysLeuGluProProTyrThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeu                               152025                                                                         CysGlnThrPheValTyrGlyGlyCysArgAlaLysSerAsnAsnPhe                               303540                                                                         LysSerAlaGluAspCysMetGluThrCysGlyGlyAla                                        455055                                                                         (2) INFORMATION FOR SEQ ID NO:78:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 508 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..499                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..331                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 332..499                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGATTAAAAGAATGAGATTTCCTTCAATTTTTACTGCAGTTTTA109                           MetArgPheProSerIlePheThrAlaValLeu                                              85-80-75                                                                       TTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAA157                            PheAlaAlaSerSerAlaLeuAlaAlaProValAsnThrThrThrGlu                               70-65-60                                                                       GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGAT205                            AspGluThrAlaGlnIleProAlaGluAlaValIleGlyTyrSerAsp                               55-50- 45                                                                      TTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACA253                            LeuGluGlyAspPheAspValAlaValLeuProPheSerAsnSerThr                               40-35-30                                                                       AATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCT301                            AsnAsnGlyLeuLeuPheIleAsnThrThrIleAlaSerIleAlaAla                               25-20-15                                                                       AAAGAAGAAGGGGTATCTTTGGATAAAAGAGATTTCTGTTTGGAACCT349                            LysGluGluGlyValSerLeuAspLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACAAC397                            ProTyrThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsn                               101520                                                                         GCCAAGGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCT445                            AlaLysAlaGlyLeuCysGlnThrPheValTyrGlyGlyCysArgAla                               253035                                                                         AAGGAAAACAACTTCAAGTCTGCTGAAGACTGCATGGAAACTTGTGGT493                            LysGluAsnAsnPheLysSerAlaGluAspCysMetGluThrCysGly                               404550                                                                         GGTGCCTAATCTAGA508                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:79:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 141 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                       MetArgPheProSerIlePheThrAlaValLeuPheAlaAlaSerSer                               85-80-75-70                                                                    AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                               65-60-55                                                                       IleProAlaGluAlaValIleGlyTyrSerAspLeuGluGlyAspPhe                               50-45- 40                                                                      AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                               35-30-25                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                               20-15-10                                                                       SerLeuAspLysArgAspPheCysLeuGluProProTyrThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAsnAlaLysAlaGlyLeu                               152025                                                                         CysGlnThrPheValTyrGlyGlyCysArgAlaLysGluAsnAsnPhe                               303540                                                                         LysSerAlaGluAspCysMetGluThrCysGlyGlyAla                                        455055                                                                         (2) INFORMATION FOR SEQ ID NO:80:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 508 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..499                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..331                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 332..499                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGATTAAAAGAATGAGATTTCCTTCAATTTTTACTGCAGTTTTA109                           MetArgPheProSerIlePheThrAlaValLeu                                              85-80-75                                                                       TTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAA157                            PheAlaAlaSerSerAlaLeuAlaAlaProValAsnThrThrThrGlu                               70-65-60                                                                       GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGAT205                            AspGluThrAlaGlnIleProAlaGluAlaValIleGlyTyrSerAsp                               55-50- 45                                                                      TTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACA253                            LeuGluGlyAspPheAspValAlaValLeuProPheSerAsnSerThr                               40-35-30                                                                       AATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCT301                            AsnAsnGlyLeuLeuPheIleAsnThrThrIleAlaSerIleAlaAla                               25-20-15                                                                       AAAGAAGAAGGGGTATCTTTGGATAAAAGAGATTTCTGTTTGGAACCT349                            LysGluGluGlyValSerLeuAspLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACAAC397                            ProTyrThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsn                               101520                                                                         GCCGAAGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCT445                            AlaGluAlaGlyLeuCysGlnThrPheValTyrGlyGlyCysArgAla                               253035                                                                         AAGTCCAACAACTTCAAGTCTGCTGAAGACTGCATGGAAACTTGTGGT493                            LysSerAsnAsnPheLysSerAlaGluAspCysMetGluThrCysGly                               404550                                                                         GGTGCCTAATCTAGA508                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:81:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 141 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                       MetArgPheProSerIlePheThrAlaValLeuPheAlaAlaSerSer                               85-80-75-70                                                                    AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                               65-60-55                                                                       IleProAlaGluAlaValIleGlyTyrSerAspLeuGluGlyAspPhe                               50-45- 40                                                                      AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                               35-30-25                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                               20-15-10                                                                       SerLeuAspLysArgAspPheCysLeuGluProProTyrThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAsnAlaGluAlaGlyLeu                               152025                                                                         CysGlnThrPheValTyrGlyGlyCysArgAlaLysSerAsnAsnPhe                               303540                                                                         LysSerAlaGluAspCysMetGluThrCysGlyGlyAla                                        455055                                                                         (2) INFORMATION FOR SEQ ID NO:82:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 508 base pairs                                                     (B) TYPE: nucleic acid                                                         (C) STRANDEDNESS: single                                                       (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: cDNA                                                       (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                              (B) LOCATION: 77..499                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: sig.sub.-- peptide                                               (B) LOCATION: 77..331                                                          (ix) FEATURE:                                                                  (A) NAME/KEY: mat.sub.-- peptide                                               (B) LOCATION: 332..499                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                       GAATTCCATTCAAGAATAGTTCAAACAAGAAGATTACAAACTATCAATTTCATACACAAT60                 ATAAACGATTAAAAGAATGAGATTTCCTTCAATTTTTACTGCAGTTTTA109                           MetArgPheProSerIlePheThrAlaValLeu                                              85-80-75                                                                       TTCGCAGCATCCTCCGCATTAGCTGCTCCAGTCAACACTACAACAGAA157                            PheAlaAlaSerSerAlaLeuAlaAlaProValAsnThrThrThrGlu                               70-65-60                                                                       GATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTACTCAGAT205                            AspGluThrAlaGlnIleProAlaGluAlaValIleGlyTyrSerAsp                               55-50- 45                                                                      TTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACA253                            LeuGluGlyAspPheAspValAlaValLeuProPheSerAsnSerThr                               40-35-30                                                                       AATAACGGGTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCT301                            AsnAsnGlyLeuLeuPheIleAsnThrThrIleAlaSerIleAlaAla                               25-20-15                                                                       AAAGAAGAAGGGGTATCTTTGGATAAAAGAGATTTCTGTTTGGAACCT349                            LysGluGluGlyValSerLeuAspLysArgAspPheCysLeuGluPro                               10-515                                                                         CCATACACTGGTCCATGTAAAGCTAGAATCATCAGATACTTCTACAAC397                            ProTyrThrGlyProCysLysAlaArgIleIleArgTyrPheTyrAsn                               101520                                                                         GCCGAAGCTGGTTTGTGTCAAACTTTCGTTTACGGTGGCTGCAGAGCT445                            AlaGluAlaGlyLeuCysGlnThrPheValTyrGlyGlyCysArgAla                               253035                                                                         AAGGAAAACAACTTCAAGTCTGCTGAAGACTGCATGGAAACTTGTGGT493                            LysGluAsnAsnPheLysSerAlaGluAspCysMetGluThrCysGly                               404550                                                                         GGTGCCTAATCTAGA508                                                             GlyAla                                                                         55                                                                             (2) INFORMATION FOR SEQ ID NO:83:                                              (i) SEQUENCE CHARACTERISTICS:                                                  (A) LENGTH: 141 amino acids                                                    (B) TYPE: amino acid                                                           (D) TOPOLOGY: linear                                                           (ii) MOLECULE TYPE: protein                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                       MetArgPheProSerIlePheThrAlaValLeuPheAlaAlaSerSer                               85-80-75-70                                                                    AlaLeuAlaAlaProValAsnThrThrThrGluAspGluThrAlaGln                               65-60-55                                                                       IleProAlaGluAlaValIleGlyTyrSerAspLeuGluGlyAspPhe                               50-45- 40                                                                      AspValAlaValLeuProPheSerAsnSerThrAsnAsnGlyLeuLeu                               35-30-25                                                                       PheIleAsnThrThrIleAlaSerIleAlaAlaLysGluGluGlyVal                               20-15-10                                                                       SerLeuAspLysArgAspPheCysLeuGluProProTyrThrGlyPro                               51510                                                                          CysLysAlaArgIleIleArgTyrPheTyrAsnAlaGluAlaGlyLeu                               152025                                                                         CysGlnThrPheValTyrGlyGlyCysArgAlaLysGluAsnAsnPhe                               303540                                                                         LysSerAlaGluAspCysMetGluThrCysGlyGlyAla                                        455055                                                                         __________________________________________________________________________ 

What is claimed is:
 1. An aprotinin analog having an inhibitory effect against serine protease, and reduced nephrotoxicity, reduced positive net charge, and reduced stability compared to native aprotinin having the following formula and set forth in the Sequence Listing as SEQ ID NO: 16:X₁ Asp Phe Cys Leu Glu Pro Pro X₂ Thr Gly Pro Cys Lys Ala Arg Ile Ile X₃ Tyr Phe Tyr X₄ Ala X₅ Ala Gly Leu Cys X₆ Thr Phe Val Tyr Gly Gly Cys Arg X₇ X₈ X₉ Asn X₁₀ Phe X₁₁ Ser Ala Glu Asp Cys Met X₁₂ Thr Cys Gly Gly Alawherein X₁ is a dipeptide selected from the group consisting of Arg-Pro, Glu-Pro, Asp-Pro, Ala-Pro, Ile-Pro, Thr-Pro, His-Pro, Leu-Pro, Gly-Pro and Ser-Pro, Pro or a hydrogen, X₂ is an amino acid residue selected from the group consisting of Tyr, Glu, Asp, Ser, Thr, Ala and Val, X₃ is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Leu, Ser, Ala, Gln and Thr, X₄ is an amino acid residue selected from the group consisting of Asn, Glu and Asp, X₅ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Thr, Val, Ala, Ser, Phe, Gln and Gly, X₆ is an amino acid residue selected from the group consisting of Gln, Glu, Asp, Val and Ala, X₇ is an amino acid residue selected from the group consisting of Ala, Asp, Glu and Gly, X₈ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Asn, Ser, Thr and Ala, X₉ is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Ser, Asn, Leu, Gly, Gln, Met and Thr, X₁₀ is an amino acid residue selected from the group consisting of Asn, Glu and Asp, X₁₁ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Leu, Tyr, Ala, Val, Thr, Ser, Pro, His and Ile, and X₁₂ is an amino acid residue selected from the group consisting of Arg, Glu, ASp, Gln, Ala, Asn, His, Gly, Ser and Thr,with the proviso that at least one of the amino acid residues X₁ -X₁₂ is different from the corresponding amino acid residue of native aprotinin.
 2. An aprotinin analog having an inhibitory effect against serine protease, and reduced nephrotoxicity, reduced positive net charge, and reduced stability compared to native aprotinin having the following formula set forth in the Sequence Listing as SEQ ID NO: 17:X₁ Asp Phe Cys Leu Glu Pro Pro X₂ Thr Gly Pro Cys X₁₃ X₁₄ X¹⁵ X₁₆ X₁₇ X₃ Tyr Phe Tyr X₄ Ala X₅ Ala Gly Leu Cys X₆ Thr Phe X₁₈ Tyr X₁₉ Gly Cys X₂₀ X₇ X₈ X₉ Asn X₁₀ Phe X₁₁ Ser Ala Glu Asp Cys Met X₁₂ Thr Cys Gly Gly Alawherein, X₁ is a dipeptide selected from the group consisting of Arg-Pro, Glu-Pro, Asp-Pro, Ala-Pro, Ile-Pro, Thr-Pro, His-Pro, Leu-Pro, Gly-Pro and Ser-Pro, Pro or a hydrogen, X₂ is an amino acid residue selected from the group consisting of Tyr, Glu, Asp, Ser, Thr, Ala and Val, X₃ is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Leu, Ser, Ala, Gln and Thr, X₄ is an amino acid residue selected from the group consisting of Asn, Glu and Asp, X₅ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Thr, Val, Ala, Ser, Phe, Gln and Gly, X₆ is an amino acid residue selected from the group consisting of Gln, Glu, Asp, Val and Ala, X₇ is an amino acid residue selected from the group consisting of Ala, Asp, Glu and Gly, X₈ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Asn, Ser, Thr and Ala, X₉ is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Ser, Asn, Leu, Gly, Gln, Met and Thr, X₁₀ is an amino acid residue selected from the group consisting of Asn, Glu and Asp, X₁₁ is an amino acid residue selected from the group consisting of Lys, Glu, Asp, Leu, Tyr, Ala, Val, Thr, Ser, Pro, His and Ile, and X₁₂ is an amino acid residue selected from the group consisting of Arg, Glu, Asp, Gln, Ala, Asn, His, Gly, Ser and Thr, X₁₃ is an amino acid residue selected from the group consisting of Lys, Arg, Glu, Leu, Met, Tyr and Phe, X₁₄ is an amino acid residue selected from the group consisting of Ala and Gly, X₁₅ is an amino acid residue selected from the group consisting of Arg, Ala, Gly, Lys, Leu, Met, Phe, Tyr, Ile and Asn, X₁₆ is an amino acid residue selected from the group consisting of Ile, Met, Leu, Phe, Thr and Glu, X₁₇ is an amino acid residue selected from the group consisting of Ile, Leu, Lys, Gln, Glu, Ser, Arg, Thr and Asn, X₁₈ is an amino acid residue selected from the group consisting of Val, Thr, Leu, Ser, Tyr, Gln, His, Pro, Phe, Asn, Ile and Lys, X₁₉ is an amino acid residue selected from the group consisting of Gly, Thr, and Ser, and X₂₀ is an amino acid residue selected from the group consisting of Gln, Lys, Met, Asn, Leu, Gly and Glu,with the proviso that at least one of the amino acid residues X₁ -X₁₂ and at least one of the amino acid residues X₁₃ -X₂₀ are different from the corresponding amino acid residue of native aprotinin.
 3. The aprotinin analog according to claim 1 or 2, wherein X₁ is Glu-Pro, X₅ is Glu, X₈ is Glu, X₁₁ is Glu, and X₂, X₃, X₄, X₆, X₇, X₉, X₁₀ and X₁₂ are as in the native aprotinin sequence.
 4. The aprotinin analog according to claim 1 or 2, wherein X₁ is Glu-Pro, X₉ is Glu, X₁₁ is Glu, and X₂, X₃, X₄, X₆, X₇, X₈, X₁₀ and X₁₂ are as in the native aprotinin sequence.
 5. The aprotinin analog according to claim 1 or 2, wherein X₉ is Glu, X₁₁ is Glu, and X₁, X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₂ are as in the native aprotinin sequence.
 6. The aprotinin analog according to claim 1 or 2, wherein X₂ is Ser, X₄ is Asp, X₅ is Thr, X₆ is Glu, X₈ is Asn, X₁₂ is Glu, and X₁, X₃, X₇, X₉, X₁₀, and X₁₁ are as in the native aprotinin sequence.
 7. The aprotinin analog according to claim 1 or 2, wherein X₂ is Ser, X₃ is Leu, X₇ is Gly, X₈ is Asn, X₉ is Gly, X₁₀ is Gln, X₁₁ is Tyr, and X₁, X₄, X₅, X₆, and X₁₂ are as in the native aprotinin sequence.
 8. The aprotinin analog according to claim 1 or 2, wherein X₁ is a peptide bond, X₉ is Ser, X₁₁ is Glu, and X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₂ are as in the native aprotinin sequence.
 9. The aprotinin analog according to claim 1 or 2, wherein and X₁ is a hydrogen, X₉ is Ser, X₁₁ is Ala, and X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₂ are as in the native aprotinin sequence.
 10. The aprotinin analog according to claim 1 or 2, wherein X₁ is a hydrogen, X₂ is Ser, X₄ is Asp, X₅ is Thr, X₆ is Glu, X₈ is Asn, X₁₂ is Glu, and X₃, X₇, X₉, X₁₀ and X₁₁ are as in the native aprotinin sequence.
 11. The aprotinin analog according to claim 1 or 2, wherein X₁ is a hydrogen, X₄ is Asp, X₅ is Thr, X₆ is Glu, X₁₂ is Glu, and X₂, X₃, X₇, X₈, X₉, X₁₀ and X₁₁ are as in the native aprotinin sequence.
 12. The aprotinin analog according to claim 1 or 2, wherein X₁ is a hydrogen, X₂ is Ser, X₇ is Gly, X₈ is Asn, X₉ is Gly, X₁₂ is Glu, and X₃, X₄, X₅, X₆, X₁₀ and X₁₁ are as in the native aprotinin sequence.
 13. The aprotinin analog according to claim 1 or 2, wherein X₁ is a hydrogen, X₉ is Ser, X₁₂ is Glu, and X₂, X₃, X₄, X₅, X₆, X₇, X₉, X₁₀ and X₁₁ are as in the native aprotinin sequence.
 14. The aprotinin analog according to claim 1 or 2, wherein X₁ is a hydrogen, X₉ is Glu, X₁₂ is Glu, and X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₁ are as in the native aprotinin sequence.
 15. The aprotinin analog according to claim 1 or 2, wherein X₁ is a hydrogen, X₅ is Glu, X₉ is Ser, X₁₂ is Glu, and X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₁ are as in the native aprotinin sequence.
 16. The aprotinin analog according to claim 1 or 2, wherein X₁ is a peptide bond, X₅ is Glu, X₉ is Glu, X₁₂ is Glu, and X₂, X₃, X₄, X₅, X₆, X₇, X₈, X₁₀ and X₁₁ are as in the native aprotinin sequence.
 17. A pharmaceutical composition comprising an aprotinin analog according to claim 1 together with a pharmaceutically acceptable carrier or excipient.
 18. A pharmaceutical composition comprising an aprotinin analog according to claim 2 together with a pharmaceutically acceptable carrier or excipient. 